Modified 5&#39; diphosphate nucleosides and oligomeric compounds prepared therefrom

ABSTRACT

Provided herein are modified 5′ diphosphate nucleosides and oligomeric compounds prepared therefrom. More particularly, modified 5′ diphosphate nucleosides are provided that can be further modified at the 2′ and 5′ positions. In some embodiments, the oligomeric compounds provided herein are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. The oligomeric compounds are also expected to be useful as primers and probes in diagnostic applications.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support undercontract #5R44GM076793-03 awarded by the NIH. The United StatesGovernment has certain rights in the invention.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled20110426_CHEM0063WOSEQ.txt, created Apr. 26, 2011, which is 5.17 Kb insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are modified 5′ diphosphate nucleosides and oligomericcompounds prepared therefrom. More particularly, the modified 5′diphosphate nucleosides provided herein are linked to the terminus of anoligomeric compound, preferably at the 5′ terminus. In certainembodiments, the oligomeric compounds provided herein are expected tohave enhanced nuclease stability. In certain embodiments, the oligomericcompounds provided herein are expected to hybridize to a portion of atarget RNA resulting in loss of normal function of the target RNA. Theoligomeric compounds provided herein are also expected to be useful asprimers and probes in diagnostic applications.

BACKGROUND OF THE INVENTION

Targeting disease-causing gene sequences was first suggested more thanthirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), andantisense activity was demonstrated in cell culture more than a decadelater (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75,280-284). One advantage of antisense technology in the treatment of adisease or condition that stems from a disease-causing gene is that itis a direct genetic approach that has the ability to modulate (increaseor decrease) the expression of specific disease-causing genes. Anotheradvantage is that validation of a therapeutic target using antisensecompounds results in direct and immediate discovery of the drugcandidate; the antisense compound is the potential therapeutic agent.

Generally, the principle behind antisense technology is that anantisense compound hybridizes to a target nucleic acid and modulatesgene expression activities or function, such as transcription ortranslation. The modulation of gene expression can be achieved by, forexample, target degradation or occupancy-based inhibition. An example ofmodulation of RNA target function by degradation is RNase H-baseddegradation of the target RNA upon hybridization with a DNA-likeantisense compound. Another example of modulation of gene expression bytarget degradation is RNA interference (RNAi). RNAi generally refers toantisense-mediated gene silencing involving the introduction of dsRNAleading to the sequence-specific reduction of targeted endogenous mRNAlevels. An additional example of modulation of RNA target function by anoccupancy-based mechanism is modulation of microRNA function. MicroRNAsare small non-coding RNAs that regulate the expression of protein-codingRNAs. The binding of an antisense compound to a microRNA prevents thatmicroRNA from binding to its messenger RNA targets, and thus interfereswith the function of the microRNA. Regardless of the specific mechanism,this sequence-specificity makes antisense compounds extremely attractiveas tools for target validation and gene functionalization, as well astherapeutics to selectively modulate the expression of genes involved inthe pathogenesis of malignancies and other diseases.

Antisense technology is an effective means for reducing the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides are routinely used forincorporation into antisense compounds to enhance one or moreproperties, such as nuclease resistance, pharmacokinetics or affinityfor a target RNA. In 1998, the antisense compound, Vitravene®(fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.)was the first antisense drug to achieve marketing clearance from theU.S. Food and Drug Administration (FDA), and is currently a treatment ofcytomegalovirus (CMV)-induced retinitis in AIDS patients.

New chemical modifications have improved the potency and efficacy ofantisense compounds, uncovering the potential for oral delivery as wellas enhancing subcutaneous administration, decreasing potential for sideeffects, and leading to improvements in patient convenience. Chemicalmodifications increasing potency of antisense compounds allowadministration of lower doses, which reduces the potential for toxicity,as well as decreasing overall cost of therapy. Modifications increasingthe resistance to degradation result in slower clearance from the body,allowing for less frequent dosing. Different types of chemicalmodifications can be combined in one compound to further optimize thecompound's efficacy.

The synthesis of 5′-substituted DNA and RNA derivatives and theirincorporation into oligomeric compounds has been reported in theliterature (Saha et al., J. Org. Chem., 1995, 60, 788-789; Wang et al.,Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; andMikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343;Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., HelveticaChimica Acta, 2004, 87, 3004-3020). The 5′-substituted monomers havealso been made as the monophosphate with modified bases (Wang et al.,Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).

A genus of modified nucleosides including optional modification at aplurality of positions including the 5′-position and the 2′-position ofthe sugar ring and oligomeric compounds incorporating these modifiednucleosides therein has been reported (see International ApplicationNumber: PCT/US94/02993, Published on Oct. 13, 1994 as WO 94/22890).

The synthesis of 5′-CH₂ substituted 2′-O-protected nucleosides and theirincorporation into oligomers has been previously reported (see Wu etal., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al.Bioconjugate Chem. 1999, 10, 921-924).

Amide linked nucleoside dimers have been prepared for incorporation intooligonucleotides wherein the 3′ linked nucleoside in the dimer (5′ to3′) comprises a 2′-OCH₃ and a 5′-(S)—CH₃ (Mesmaeker et al., Synlett,1997, 1287-1290).

A genus of 2′-substituted 5′-CH₂ (or O) modified nucleosides and adiscussion of incorporating them into oligonucleotides has beenpreviously reported (see International Application Number:PCT/US92/01020, published on Feb. 7, 1992 as WO 92/13869).

The synthesis of modified 5′-methylene phosphonate monomers having2′-substitution and their use to make modified antiviral dimers has beenpreviously reported (see U.S. patent application Ser. No. 10/418,662,published on Apr. 6, 2006 as US 2006/0074035).

The synthesis of 5′-methylenebis(phosphonate) monomers having a 2′-OCH₃(or OH) and their use to make dinucleotide mRNA cap analogs have beenpreviously described (see Rydizk et al., Org. Biomol. Chem., 2009, 7,4763-4776 and Myers et al., J. Org. Chem., 1965, 30, 1517-1520). Othernucleosides comprising 5′-methylene or 5′-difluoromethylenebisphosphonates have also been reported wherein the hydroxyl groups atthe 2′ or/and 3′ positions are protected or substituted (see publishedInternational Applications WO 94/29331, WO 98/15563 and WO 2006/038865).

A genus of modified 5′-diphosphate and 5′-triphosphate nucleosides andtheir potential use as antiviral agents has been described (seepublished International Application WO 03/072757). Other modified5′-diphosphate mononucleosides and their use to make dimeric compoundshave been previously reported (see published International ApplicationWO 2007/020018 and U.S. Pat. Nos. 7,018,985, issued on Mar. 28, 2006;7,101,860, issued on Sep. 5, 2006 and 7,132,408, issued on Nov. 7,2006).

The synthesis of 5′(S)- and 5′(R)-methyl (or propyl) ribonucleosideshaving a 5′-pyro-phosphate group (P—O—P) have been described along withtheir physico-chemical and biological property studies (see Kappler etal., J. Med. Chem., 1982, 25, 1179-1184; Hai et al., J. Med. Chem.,1982, 25, 1184-1188; David et al., Journal of Chemical Society PerkinTrans I: Org. Bioorg. Chem., (1972-1999) (1982), 2, 385-393 andMikhailov et al., Nucleosides Nucleotides, 1991, 10, 339-343).

Various 5′-monophosphate and 5′-triphosphate nucleosides having a5′-acylaminomethyl group (5′-CH₂NH(C═O)CH₃) (and analogs thereof) havebeen reported in the literature and were further evaluated forbiological activities (see Hampton et al., J. Med. Chem., 1979, 22,621-631 and J. Med. Chem., 1976, 19, 1371-1377; Kappler et al., J. Org.Chem., 1975, 40, 1378-1385, J. Med. Chem., 1987, 30, 1599-1603 and J.Med. Chem., 1990, 33, 2545-2551).

The synthesis of 5′-monophosphate and 5′-triphosphate DNA and RNAmonomers, oligomers comprising these monomers, Tm evaluations of theseoligomers and their preparation as fluorescent probes have beendescribed (see PCT International Application WO 00/14101, published onMar. 16, 2000).

The preparation of deoxyribonucleosides and ribonucleosides comprising a5′-imidodiphos-phate group (P—NH—P) and their biological evaluationshave been previously reported (see Tomasz et al., Nucleic AcidsResearch, 1988, 16, 8645-8664).

BRIEF SUMMARY OF THE INVENTION

Provided herein are modified 5′ diphosphate nucleosides and oligomericcompounds prepared therefrom. More particularly, the modified 5′diphosphate nucleosides as provided herein are linked to the terminus ofan oligomeric compound, preferably at the 5′ terminus. In certainembodiments, the oligomeric compounds provided herein are expected tohave enhanced nuclease stability. In certain embodiments, the oligomericcompounds and compositions provided herein that incorporate thesemodified nucleosides are expected to hybridize to a portion of a targetRNA resulting in loss of normal function of the target RNA. Theoligomeric compounds are also expected to be useful as primers andprobes in diagnostic applications.

The variables are defined individually in further detail herein. It isto be understood that the modified nucleosides and oligomeric compoundsprovided herein include all combinations of the embodiments disclosedand variables defined herein.

In certain embodiments, compounds are provided herein having Formula I:

wherein:

Bx is a heterocyclic base moiety;

each Pg is a hydroxyl protecting group;

M₁ is H, OH or OR₁;

M₂ is OH, OR₁ or N(R₁)(R₂);

each R₁ and R₂ is, independently, alkyl or substituted alkyl;

r is 0 or 1;

A is O, S, CR₃R₄ or N(R₅);

R₃ and R₄ are each, independently H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

R₅ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or a protecting group;

Q₁ and Q₂ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₆)(R₇)]_(n)-[(C═O)_(m)—X]_(j)—Z;

each R₆ and R₇ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, 1-1, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from H, halogen, OJ₁,N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂), C(=L)N(J₁)(J₂),C(=L)N(H)—(CH₂)₂N(J₁)(J₂) or a mono or poly cyclic ring system;

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

when Q₁ and Q2 are each H and G is H or OH then A is other than O.

In certain embodiments, Bx is a pyrimidine, substituted pyrimidine,purine or substituted purine. In certain embodiments, Bx is uracil,5-thiazolo-uracil, thymine, cytosine, 5-methylcytosine,5-thiazolo-cytosine, adenine, guanine or 2,6-diaminopurine. In certainembodiments, Bx is uracil, thymine, cytosine, 5-methylcytosine, adenineor guanine.

In certain embodiments, r is 1, M₁ is H and M₂ is OH. In certainembodiments, r is 0, M₁ is O(CH₂)₂CN and M₂ is N[CH(CH₃)₂]₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃,O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃,O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₆)(R₇), O(CH₂)₂—ON(R₆)(R₇),O(CH₂)₂—O(CH₂)₂—N(R₆)(R₇), OCH₂C(═O)—N(R₆)(R₇),OCH₂C(═O)—NR₈)—(CH₂)₂—N(R₆)(R₇) or O(CH₂)₂—N(R₈)—C(═NR₉)[N(R₆)(R₇)]wherein R₆, R₇, R₈ and R₉ are each, independently, H or C₁-C₆ alkyl. Incertain embodiments, G is halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃,OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certainembodiments, G is F, OCH₃, O(CH₂)₂—OCH₃, OCH₂C(═O)—N(H)CH₃ orOCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂. In certain embodiments, G isO(CH₂)₂—OCH₃. In certain embodiments, G is F.

In certain embodiments, each Pg is, independently, methyl, ethyl,isopropyl, tert-butyl, 2-cyanoethyl, benzyl, phenyl, 4-methoxybenzyl,4-chlorobenzyl or 2-chlorophenyl

In certain embodiments, each Pg is CH₂CH₃ or benzyl.

In certain embodiments, Q₁ and Q₂ are each H. In certain embodiments,one of Q₁ and Q₂ is H and the other of Q₁ and Q₂ is C₁-C₆ alkyl. Incertain embodiments, the other of Q₁ and Q₂ is CH₃. In certainembodiments, one of Q₁ and Q₂ is H and the other of Q₁ and Q₂ issubstituted C₁-C₆ alkyl. In certain embodiments, Q₁ and Q₂ are eachindependently, C₁-C₆ alkyl or substituted C₁-C₆ alkyl.

In certain embodiments, A is O. In certain embodiments, A is S.

In certain embodiments, A is N(R₅). In certain embodiments, R₅ is H or aprotecting group. In certain embodiments, R₅ is C₁-C₆ alkyl orsubstituted C₁-C₆ alkyl. In certain embodiments, R₅ is CH₃.

In certain embodiments, A is CR₃R₄. In certain embodiments, R₃ and R₄are each H. In certain embodiments, one of R₃ and R₄ is H and the otherof R₃ and R₄ is other than H. In certain embodiments, the other of R₃and R₄ is F. In certain embodiments, the other of R₃ and R₄ is C₁-C₆alkyl or substituted C₁-C₆ alkyl. In certain embodiments, the other ofR₃ and R₄ is CH₃. In certain embodiments, R₃ and R₄ are each other thanH.

In certain embodiments, compounds are provided herein having Formula Ia:

wherein:

Bx is a heterocyclic base moiety;

each Pg is a hydroxyl protecting group;

M₁ is H, OH or OR₁;

M₂ is OH, OR₁ or N(R₁)(R₂);

each R₁ and R₂ is, independently, alkyl or substituted alkyl;

r is 0 or 1;

A is O, S, CR₃R₄ or N(R₅);

R₃ and R₄ are each, independently H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

R₅ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or a protecting group;

Q₁ and Q₂ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₆)(R₇)]_(n)—[(C═O)_(m)—X]_(j)—Z;

each R₆ and R₇ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from H, halogen, OJ₁,N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)N(J₁)(J₂), C(=L)N(J₁)(J₂),C(=L)N(H)—(CH₂)₂N(J₁)(J₂) or a mono or poly cyclic ring system;

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

when Q₁ and Q₂ are each H and G is H or OH then A is other than O.

In certain embodiments, oligomeric compounds are provided wherein eacholigomeric compound comprises a compound having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T₁ is an internucleoside linking group linking the compound of FormulaII to the remainder of the oligomeric compound;

each M₃ is, independently, H or a hydroxyl protecting group;

A is O, S, CR₃R₄ or N(R₅);

R₃ and R₄ are each, independently H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

R₅ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or a protecting group;

Q₁ and Q₂ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₆)(R₇)]_(n)—[(C═O)_(m)—X]_(j)—Z;

each R₆ and R₇ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from H, halogen, OJ₁,N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂), C(=L)N(J₁)(J₂),C(=L)N(H)—(CH₂)₂N(J₁)(J₂) or a mono or poly cyclic ring system;

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

when Q₁ and Q₂ are each H and G is H or OH then A is other than O.

In certain embodiments, Bx is a pyrimidine, substituted pyrimidine,purine or substituted purine. In certain embodiments, Bx is uracil,5-thiazolo-uracil, thymine, cytosine, 5-methylcytosine,5-thiazolo-cytosine, adenine, guanine or 2,6-diaminopurine. Bx isuracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃,O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃,O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₆)(R₇), O(CH₂)₂—ON(R₆)(R₇),O(CH₂)₂—O(CH₂)₂—N(R₆)(R₇), OCH₂C(═O)—N(R₆)(R₇),OCH₂C(═O)—N(R₈)—(CH₂)₂—N(R₆)(R₇) or O(CH₂)₂—N(R₈)—C(═NR₉)[N(R₆)(R₇)]wherein R₆, R₇, R₈ and R₉ are each, independently, H or C₁-C₆ alkyl. Incertain embodiments, G is halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃,OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certainembodiments, G is F, OCH₃, O(CH₂)₂—OCH₃, OCH₂C(═O)—N(H)CH₃ orOCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂. In certain embodiments, G isO(CH₂)₂—OCH₃. In certain embodiments, G is F.

In certain embodiments, each M₃ is, independently, methyl, ethyl,isopropyl, tert-butyl, 2-cyanoethyl, benzyl, phenyl, 4-methoxybenzyl,4-chlorobenzyl or 2-chlorophenyl.

In certain embodiments, each M₃ is, independently, CH₂CH₃ or benzyl.

In certain embodiments, Q₁ and Q₂ are each H. In certain embodiments,one of Q₁ and Q₂ is H and the other of Q₁ and Q₂ is C₁-C₆ alkyl. Incertain embodiments, the other one of Q₁ and Q₂ is CH₃. In certainembodiments, one of Q₁ and Q₂ is H and the other of Q₁ and Q₂ issubstituted C₁-C₆ alkyl. In certain embodiments, Q₁ and Q₂ are eachindependently, C₁-C₆ alkyl or substituted C₁-C₆ alkyl.

In certain embodiments, A is O. In certain embodiments, A is S.

In certain embodiments, A is N(R₅). In certain embodiments, R₅ is H or aprotecting group. In certain embodiments, R₅ is C₁-C₆ alkyl orsubstituted C₁-C₆ alkyl. In certain embodiments, R₅ is CH₃.

In certain embodiments, A is CR₃R₄. In certain embodiments, R₃ and R₄are each H. In certain embodiments, one of R₃ and R₄ is H and the otherof R₃ and R₄ is other than H. In certain embodiments, the other of R₃and R₄ is F. In certain embodiments, the other of R₃ and R₄ is C₁-C₆alkyl or substituted C₁-C₆ alkyl. In certain embodiments, the other ofR₃ and R₄ is CH₃. In certain embodiments, R₃ and R₄ are each other thanH.

In certain embodiments, oligomeric compounds are provided wherein eacholigomeric compound comprises a compound having Formula IIa:

wherein:

Bx is a heterocyclic base moiety;

T₁ is an internucleoside linking group linking the compound of FormulaIIa to the remainder of the oligomeric compound;

each M₃ is, independently, H or a hydroxyl protecting group;

A is O, S, CR₃R₄ or N(R₅);

R₃ and R₄ are each, independently H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

R₅ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or a protecting group;

Q₁ and Q₂ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₆)(R₇)]_(n)—[(C═O)_(m)—X]_(j)—Z;

each R₆ and R₇ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from H, halogen, OJ₁,N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂), C(=L)N(J₁)(J₂),C(=L)N(H)—(CH₂)₂N(J₁)(J₂) or a mono or poly cyclic ring system;

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

when Q₁ and Q₂ are each H and G is H or OH then A is other than 0.

In certain embodiments, the invention provides an oligomeric compoundwherein the 5′-terminal nucleoside has formula II or IIa.

In certain such embodiments, the remainder of the oligomeric compoundcomprises at least one modified nucleoside. In certain embodiments, theoligomeric compound comprises a modified base. In certain embodiments,the oligomeric compound comprises a sugar surrogate. In certain suchembodiments, the sugar surrogate is a tetrahydropyran. In certainembodiments, the tetrahydropyran is F—HNA.

In certain embodiments, the remainder of the oligomeric compoundcomprises at least one nucleoside comprising a modified sugar. Incertain embodiments, the at least one modified nucleoside comprising amodified sugar is selected from a bicyclic nucleoside and a 2′-modifiednucleoside. In certain embodiments, the at least one modified nucleosideis a bicyclic nucleoside. In certain embodiments, the bicyclicnucleoside is a (4′-CH₂—O-2′) BNA nucleoside. In certain embodiments,the bicyclic nucleoside is a (4′-(CH₂)₂—O-2′) BNA nucleoside. In certainembodiments, the bicyclic nucleoside is a (4′-C(CH₃)H—O-2′) BNAnucleoside. In certain embodiments, the at least one modified nucleosideis a 2′-modified nucleoside. In certain embodiments, the at least one2′-modified nucleoside is selected from a 2′-F nucleoside, a 2′-OCH₃nucleoside, and a 2′-O(CH₂)₂OCH₃ nucleoside. In certain embodiments, theat least one 2′-modified nucleoside is a 2′-F nucleoside. In certainembodiments, the at least one 2′-modified nucleoside is a 2% OCH₃nucleoside. In certain embodiments, the at least one 2′-modifiednucleoside is a 2′-O(CH₂)₂OCH₃ nucleoside.

In certain embodiments, the remainder of the oligomeric compoundcomprises at least one unmodified nucleoside. In certain embodiments,the unmodified nucleoside is a ribonucleoside. In certain embodiments,the unmodified nucleoside is a deoxyribonucleoside.

In certain embodiments, the remainder of the oligomeric compoundcomprises at least two modified nucleosides. In certain embodiments, theat least two modified nucleosides comprise the same modification. Incertain embodiments, the at least two modified nucleosides comprisedifferent modifications. In certain embodiments, at least one of the atleast two modified nucleosides comprises a sugar surrogate. In certainembodiments, at least one of the at least two modified nucleosidescomprises a 2′-modification. In certain embodiments, each of the atleast two modified nucleosides is independently selected from 2′-Fnucleosides, 2′-OCH₃ nucleosides and 2′-O(CH₂)₂OCH₃ nucleosides. Incertain embodiments, each of the at least two modified nucleosides is a2′-F nucleoside. In certain embodiments, each of the at least twomodified nucleosides is a 2′-OCH₃ nucleosides. In certain embodiments,each of the at least two modified nucleosides is a 2′-O(CH₂)₂OCH₃nucleoside. In certain embodiments, essentially every nucleoside of theoligomeric compound is a modified nucleoside. In certain embodiments,every nucleoside of the oligomeric compound is a modified nucleoside.

In certain embodiments, the remainder of the oligomeric compoundcomprises:

-   -   1-20 first-type regions, each first-type region independently        comprising 1-20 contiguous nucleosides wherein each nucleoside        of each first-type region comprises a first-type modification;    -   0-20 second-type regions, each second-type region independently        comprising 1-20 contiguous nucleosides wherein each nucleoside        of each second-type region comprises a second-type modification;        and    -   0-20 third-type regions, each third-type region independently        comprising 1-20 contiguous nucleosides wherein each nucleoside        of each third-type region comprises a third-type modification;        wherein    -   the first-type modification, the second-type modification, and        the third-type modification are each independently selected from        2′-F, 2′-OCH₃, 2′-O(CH₂)₂OCH₃, BNA, F—HNA, 2′-H and 2′-OH;    -   provided that the first-type modification, the second-type        modification, and the third-type modification are each different        from one another.

In certain embodiments, the remainder of the oligomeric compoundcomprises 2-20 first-type regions; 3-20 first-type regions; 4-20first-type regions; 5-20 first-type regions; or 6-20 first-type regions.In certain embodiments, the remainder of the oligomeric compoundcomprises 1-20 second-type regions; 2-20 second-type regions; 3-20second-type regions; 4-20 second-type regions; or 5-20 second-typeregions. In certain embodiments, the remainder of the oligomericcompound comprises 1-20 third-type regions; 2-20 third-type regions;3-20 third-type regions; 4-20 third-type regions; or 5-20 third-typeregions.

In certain embodiments, the remainder of the oligomeric compoundcomprises a third-type region at the 3′-end of the oligomeric compound.In certain embodiments, the remainder of the oligomeric compoundcomprises a third-type region at the 3′-end of the oligomeric compound.In certain embodiments, the third-type region contains from 1 to 3modified nucleosides and the third-type modification is 2′-O(CH₂)₂OCH₃.In certain embodiments, the third same type region contains two modifiednucleosides and the third-type modification is 2′-O(CH₂)₂OCH₃.

In certain embodiments, each first-type region contains from 1 to 5modified nucleosides. In certain embodiments, each first-type regioncontains from 6 to 10 modified nucleosides. In certain embodiments, eachfirst-type region contains from 11 to 15 modified nucleosides. Incertain embodiments, each first-type region contains from 16 to 20modified nucleosides.

In certain embodiments, the first-type modification is 2′-F. In certainembodiments, the first-type modification is 2′-OMe. In certainembodiments, the first-type modification is DNA. In certain embodiments,the first-type modification is 2′-O(CH₂)₂OCH₃. In certain embodiments,the first-type modification is 4′-CH₂—O-2′. In certain embodiments, thefirst-type modification is 4′-(CH₂)₂—O-2′. In certain embodiments, thefirst-type modification is 4′-C(CH₃)H—O-2′. In certain embodiments, eachsecond-type region contains from 1 to 5 modified nucleosides. In certainembodiments, each second-type region contains from 6 to 10 modifiednucleosides. In certain embodiments, each second-type region containsfrom 11 to 15 modified nucleosides. In certain embodiments, eachsecond-type region contains from 16 to 20 modified nucleosides. Incertain embodiments, the second-type modification is 2′-F. In certainembodiments, the second-type modification is 2′-OMe. In certainembodiments, the second-type modification is DNA. In certainembodiments, the second-type modification is 2′-O(CH₂)₂OCH₃. In certainembodiments, the second-type modification is 4′-CH₂—O-2′. In certainembodiments, the second-type modification is 4′-(CH₂)₂—O-2′. In certainembodiments, the second-type modification is 4′-C(CH₃)H—O-2′. In certainembodiments, the oligomeric compound has an alternating motif whereinthe first-type regions alternate with the second-type regions.

In certain embodiments, the invention provides oligomeric compoundswherein the 5′ terminal nucleoside is a compound of formula II or IIaand the remainder of the oligomeric compound comprises at least oneregion of nucleosides having a nucleoside motif:

(A)_(n)-(B)_(n)-(A)_(n)-(B)_(n), wherein:

A an B are differently modified nucleosides; and

each n is independently selected from 1, 2, 3, 4, and 5.

In certain embodiments, A and B are each independently selected from abicyclic and a 2′-modified nucleoside. In certain embodiments, at leastone of A and B is a bicyclic nucleoside. In certain embodiments, atleast one of A and B is a (4′-CH₂—O-2′) BNA nucleoside. In certainembodiments, at least one of A and B is a (4′-(CH₂)₂—O-2′) BNAnucleoside. In certain embodiments, at least one of A and B is a(4′-C(CH₃)H—O-2′) BNA nucleoside. In certain embodiments, at least oneof A and B is a 2′-modified nucleoside. In certain embodiments, the2′-modified nucleoside is selected from: a 2′-F nucleoside, a 2′-OCH₃nucleoside, and a 2′-O(CH₂)₂OCH₃ nucleoside. In certain embodiments, Aand B are each independently selected from: a 2′-F nucleoside, a 2′-OCH₃nucleoside, a 2′-O(CH₂)₂OCH₃ nucleoside, a (4′-CH₂—O-2′) BNA nucleoside,a (4′-(CH₂)₂—O-2′) BNA nucleoside, a (4′-C(CH₃)H—O-2′) BNA nucleoside, aDNA nucleoside, an RNA nucleoside, and an F—HNA nucleoside. In certainembodiments, A and B are each independently selected from: a 2′-Fnucleoside, a 2′-OCH₃ nucleoside, a (4′-CH₂—O-2′) BNA nucleoside, a(4′-(CH₂)₂—O-2′) BNA nucleoside, a (4′-C(CH₃)H—O-2′) BNA nucleoside, anda DNA nucleoside. In certain embodiments, one of A and B is a 2′-Fnucleoside. In certain embodiments, one of A and B is a 2′-OCH₃nucleoside. In certain embodiments, one of A and B is a 2′-O(CH₂)₂OCH₃nucleoside. In certain embodiments, A is a 2′-F nucleoside and B is a2′-OCH₃ nucleoside. In certain embodiments, A is a 2′-OCH₃ nucleosideand B is a 2′-F nucleoside. In certain embodiments, one of A and B isselected from a (4′-CH₂—O-2′) BNA nucleoside, a (4′-(CH₂)₂—O-2′) BNAnucleoside, and a (4′-C(CH₃)H—O-2′) BNA nucleoside and the other of Aand B is a DNA nucleoside.

In certain embodiments, the invention provides oligomeric compoundswherein the 5′ terminal nucleoside is a compound of formula II or IIaand the remainder of the oligomeric compound comprises at least oneregion of nucleosides having a nucleoside motif:(A)_(x)-(B)₂-(A)_(Y)-(B)₂-(A)_(Z)-(B)₃ wherein

A is a nucleoside of a first type;

B is a nucleoside of a second type;

X is 0-10;

Y is 1-10; and

Z is 1-10.

In certain embodiments, X is selected from 0, 1, 2 and 3. In certainembodiments, X is selected from 4, 5, 6 and 7. In certain embodiments, Yis selected from 1, 2 and 3. In certain embodiments, Y is selected from4, 5, 6 and 7. In certain embodiments, Z is selected from 1, 2 and 3. Incertain embodiments, Z is selected from 4, 5, 6 and 7. In certainembodiments, A is a 2′-F nucleoside. In certain embodiments, B is a2′-OCH₃ nucleoside.

In certain embodiments, the invention provides oligomeric compoundswherein the 5′ terminal nucleoside is a compound of formula II or IIaand wherein the oligomeric compounds comprises a 3′-region consisting offrom 1 to 5 nucleosides at the 3′-end of the oligomeric compoundwherein:

the nucleosides of the 3′-region each comprises the same modification asone another; and

the nucleosides of the 3′-region are modified differently than the lastnucleoside adjacent to the 3′-region.

In certain embodiments, the modification of the 3′-region is differentfrom any of the modifications of any of the other nucleosides of theoligomeric compound. In certain embodiments, the nucleosides of the3′-region are 2′—O(CH₂)₂OCH₃ nucleosides. In certain embodiments, the3′-region consists of 2 nucleosides. In certain embodiments, the3′-region consists of 3 nucleosides. In certain embodiments, eachnucleoside of the 3′-region comprises a uracil base. In certainembodiments, each nucleoside of the 3′-region comprises an adenine base.In certain embodiments, each nucleoside of the 3′-region comprises athymine base.

In certain embodiments, the remainder of the oligomeric compoundcomprises a region of uniformly modified nucleosides. In certainembodiments, the region of uniformly modified nucleosides comprises 2-20contiguous uniformly modified nucleosides. In certain embodiments, theregion of uniformly modified nucleosides comprises 3-20 contiguousuniformly modified nucleosides. In certain embodiments, the region ofuniformly modified nucleosides comprises 4-20 contiguous uniformlymodified nucleosides. In certain embodiments, the region of uniformlymodified nucleosides comprises 5-20 contiguous uniformly modifiednucleosides. In certain embodiments, the region of uniformly modifiednucleosides comprises 6-20 contiguous uniformly modified nucleosides. Incertain embodiments, the region of uniformly modified nucleosidescomprises 5-15 contiguous uniformly modified nucleosides. In certainembodiments, the region of uniformly modified nucleosides comprises 6-15contiguous uniformly modified nucleosides. In certain embodiments, theregion of uniformly modified nucleosides comprises 5-10 contiguousuniformly modified nucleosides. In certain embodiments, the region ofuniformly modified nucleosides comprises 6-10 contiguous uniformlymodified nucleosides.

In certain embodiments, the remainder of the oligomeric compoundcomprises a region of alternating modified nucleosides and a region ofuniformly modified nucleosides. In certain embodiments, the region ofalternating nucleotides is 5′ of the region of fully modifiednucleosides. In certain embodiments, the region of alternatingnucleotides is 3′ of the region of fully modified nucleosides. Incertain embodiments, the alternating region and the fully modifiedregion are immediately adjacent to one another. In certain embodiments,the oligomeric compound has additional nucleosides between thealternating region and the fully modified region.

In certain embodiments, the remainder of the oligomeric compoundcomprises at least one region of nucleosides having a motif I:

N_(f)(PS)N_(m)(PO), wherein:

N_(f) is a 2′-F nucleoside,

N_(m) is a 2′-OCH₃ nucleoside

PS is a phosphorothioate linking group; and

PO is a phosphodiester linking group.

In certain embodiments, the 5′ terminal nucleoside is a compound offormula II or IIa and the second nucleoside from the 5′ terminal end isN_(f).

In certain embodiments, the oligomeric compound comprises at least 2, or3, or 4, or 6, or 7, or 8, or 9, or 10 separate regions having motif I.

In certain embodiments, the remainder of the oligomeric compoundcomprises at least one region having a nucleoside motif selected from:

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA; or

ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of asecond type.

In certain embodiments, oligomeric compounds of the invention compriseone or more conjugate groups. In certain embodiments, oligomericcompounds of the invention consist of an oligonucleotide.

In certain embodiments, the invention provides oligomeric compoundshaving the formula:

5′-(Z)-(L-Q₁-L-Q₂)_(t)-(L-Q₁)_(u)-(L-Q₃)_(v)-(G)_(a)-3′

wherein:

each L is an internucleoside linking group;

G is a conjugate or a linking group linking the oligomeric compound to aconjugate;

a is 0 or 1;

each of Q₁, Q₂ and Q₃ is, independently, a 2′-modified nucleoside havinga 2′-substituent group selected from halogen, allyl, amino, azido,O-allyl, O—C₁-C₆ alkyl, OCF₃, O—(CH₂)₂—O—CH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(J₅)(J₆) and O—CH₂—C(═O)—N(J₅)(J₆), where each J₅ and J₆ is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₆ alkyl; provided that Q₁, Q₂ and Q₃ are differentfrom one another;

t is from 4 to 8;

u is 0 or 1;

v is from 1 to 3; and

Z is a compound of formula II or IIa.

In certain embodiments, Q₁ and Q₂ is, independently, a 2′-modifiednucleoside having a 2′-substituent group selected from halogen andO—C₁-C₆ alkyl. In certain embodiments, each Q₁ and Q₂ is, independently,a 2′-modified nucleoside having a 2′-substituent group selected from Fand O-methyl. In certain embodiments, each Q₃ is a 2′-modifiednucleoside having a 2′-substituent group of O—(CH₂)₂—OCH₃. In certainembodiments, a is 0. In certain embodiments, v is 2. In certainembodiments, u is 0. In certain embodiments, u is 1.

In certain of any of the above embodiments, the remainder of theoligomeric compound comprises an oligonucleotide consisting of 8-80linked nucleosides; 8-26 linked nucleosides; 10-24 linked nucleosides;16-22 linked nucleosides; 16-18 linked nucleosides; or 19-22 linkednucleosides.

In certain of any of the above embodiments, the second nucleoside fromthe 5′-end comprises a sugar moiety comprising a 2′-substituent selectedfrom OH and a halogen. In certain embodiments, the second nucleosidefrom the 5′-end is a 2′-F modified nucleoside.

In certain of any of the above embodiments, the oligomeric compoundcomprises at least one modified linking group. In certain embodiments,each internucleoside linking group is, independently, phosphodiester orphosphorothioate. In certain embodiments, the 5′-most internucleosidelinking group is a phosphorothioate linking group. In certainembodiments, at least one phosphorothioate region comprising at leasttwo contiguous phosphorothioate linking groups. In certain embodiments,the at least one phosphorothioate region comprises from 3 to 12contiguous phosphorothioate linking groups. In certain embodiments, theat least one phosphorothioate region comprises from 6 to 8phosphorothioate linking groups. In certain embodiments, the at leastone phosphorothioate region is located at the 3′-end of the oligomericcompound. In certain embodiments, the at least one phosphorothioateregion is located within 3 nucleosides of the 3′-end of the oligomericcompound. In certain embodiments, the 7-9 internucleoside linkages atthe 3′ end of the oligonucleotide are phosphorothioate linkages and theinternucleoside linkage at the 5′-end is a phosphorothioate linkage.

In certain embodiments, the invention provides oligomeric compoundscomprising an oligonucleotide consisting of 10 to 30 linked nucleosideswherein:

(a) the nucleoside at the 5′ end has formula II or IIa:(b) the sugar moiety of the second nucleoside from the 5′-end isselected from an unmodified 2′-OH sugar, and a modified sugar comprisinga modification selected from: 2′-halogen, 2′O-alkyl, and2′-β-substituted alkyl; and(c) the first internucleoside linkage at the 5′-end and the last seveninternucleoside linkages at the 3′-end are phosphorothioate linkages;and(d) at least one internucleoside linkage is other than aphosphorothioate linkage.

In certain embodiments, the oligomeric compound is an antisensecompound. In certain embodiments, the antisense compound is an RNAicompound. In certain embodiments, the antisense compound is asingle-stranded RNAi compound. In certain embodiments, the antisensecompound is a double-stranded RNAi compound (siRNA) in which one or bothstrands is an oligomeric compound as disclosed herein. In certainembodiments, the antisense compound is a microRNA mimic. In certainembodiments, the antisense compound is an RNase H antisense compound. Incertain embodiments, the antisense compound modulates splicing.

In certain embodiments, at least a portion of the nucleobase sequence ofthe oligomeric compound is complementary to a portion of a targetnucleic acid, wherein the target nucleic acid is selected from: a targetmRNA, a target pre-mRNA, a target microRNA, and a target non-coding RNA.In certain embodiments, the nucleobase sequence of the oligonueleotide aregion of 100% complementarity to the target nucleic acid and whereinthe region of 100% complementarity is at least 10 nucleobases. Incertain embodiments, the region of 100% complementarity is at least 15nucleobases. In certain embodiments, the region of 100% complementarityis at least 20 nucleobases. In certain embodiments, the oligonucleotideis at least 85% complementary to the target nucleic acid. In certainembodiments, the oligonucleotide is at least 90% complementary to thetarget nucleic acid. In certain embodiments, the oligonucleotide is atleast 95% complementary to the target nucleic acid. In certainembodiments, the oligonucleotide is at least 98% complementary to thetarget nucleic acid. In certain embodiments, the oligonucleotide is 100%complementary to the target nucleic acid.

In certain embodiments, the antisense compound is a microRNA mimichaving a nucleobase sequence comprising a portion that is at least 80%identical to the seed region of a microRNA and that has overall identitywith the microRNA of at least 70%. In certain embodiments, thenucleobase sequence of the microRNA mimic has a portion that is at least80% identical to the sequence of the seed region of a microRNA and hasoverall identity with the microRNA of at least 75%. In certainembodiments, the nucleobase sequence of the microRNA mimic has a portionthat is at least 80% identical to the sequence of the seed region of amicroRNA and has overall identity with the microRNA of at least 80%. Incertain embodiments, the nucleobase sequence of the microRNA mimic has aportion that is at least 100% identical to the sequence of the seedregion of a microRNA and has overall identity with the microRNA of atleast 80%. In certain embodiments, the nucleobase sequence of themicroRNA mimic has a portion that is at least 100% identical to thesequence of the seed region of a microRNA and has overall identity withthe microRNA of at least 85%. In certain embodiments, the nucleobasesequence of the microRNA mimic has a portion that is 100% identical tothe sequence of the microRNA. In certain embodiments, nucleobasesequence of the oligonucleotide comprises a region of 100%complementarity to a seed match segment of a target nucleic acid. Incertain embodiments, the antisense compound is a microRNA mimic having anucleobase sequence comprising a portion that is at least 80% identicalto the seed region of a microRNA and that has overall identity with themicroRNA of at least 50%. In certain embodiments, the antisense compoundis a microRNA mimic having a nucleobase sequence comprising a portionthat is at least 80% identical to the seed region of a microRNA and thathas overall identity with the microRNA of at least 55%. In certainembodiments, the antisense compound is a microRNA mimic having anucleobase sequence comprising a portion that is at least 80% identicalto the seed region of a microRNA and that has overall identity with themicroRNA of at least 60%. In certain embodiments, the antisense compoundis a microRNA mimic having a nucleobase sequence comprising a portionthat is at least 80% identical to the seed region of a microRNA and thathas overall identity with the microRNA of at least 65%. In certainembodiments, the oligomeric compound comprises a nucleobase sequenceselected from a microRNA sequence found in miRBase. In certainembodiments, the oligomeric compound consists of a nucleobase sequenceselected from a microRNA sequence found in miRBase.

In certain embodiments, the target nucleic acid is a target mRNA. Incertain embodiments, the target nucleic acid is a target pre-mRNA. Incertain embodiments, the target nucleic acid is a non-coding RNA. Incertain embodiments, the target nucleic acid is a microRNA. In certainembodiments, the target nucleic acid is a pre-mir. In certainembodiments, the target nucleic acid is a pri-mir.

In certain embodiments, the nucleobase sequence of the oligonucleotidecomprises a region of 100% complementarity to the target nucleic acidand wherein the region of 100% complementarity is at least 10nucleobases. In certain embodiments, the nucleobase sequence of theoligonucleotide comprises a region of 100% complementarity to the targetnucleic acid and wherein the region of 100% complementarity is at least6 nucleobases. In certain embodiments, the nucleobase sequence of theoligonucleotide comprises a region of 100% complementarity to the targetnucleic acid and wherein the region of 100% complementarity is at least7 nucleobases. In certain embodiments, the target nucleic acid is amammalian target nucleic acid. In certain embodiments, the mammaliantarget nucleic acid is a human target nucleic acid.

In certain embodiments, oligomeric compounds comprise from 1 to 3terminal group nucleosides on at least one end of the oligonucleotide.In certain embodiments, oligomeric compound comprise from 1 to 3terminal group nucleosides at the 3′-end of the oligonucleotide. Incertain embodiments, oligomeric compound comprise from 1 to 3 terminalgroup nucleosides at the 5′-end of the oligonucleotide.

In certain embodiments, oligomeric compounds of the invention are singlestranded.

In certain embodiments, oligomeric compounds of the invention are pairedwith a second oligomeric compound to form a double stranded compound.

In certain embodiments, methods of inhibiting gene expression areprovided comprising contacting a cell with an oligomeric compound or adouble stranded composition as provided herein, wherein said oligomericcompound comprises from about 8 to about 40 monomeric subunits and iscomplementary to a target RNA. In certain embodiments, the cell is in ananimal. In certain embodiments, the cell is in a human. In certainembodiments, the target RNA is selected from mRNA, pre-mRNA and microRNA. In certain embodiments, the target RNA is mRNA. In certainembodiments, the target RNA is human mRNA. In certain embodiments, thetarget RNA is cleaved thereby inhibiting its function.

In certain embodiments, methods of inhibiting gene expression areprovided comprising contacting a cell with an oligomeric compound or adouble stranded composition as provided herein, wherein said oligomericcompound comprises from about 8 to about 40 monomeric subunits and iscomplementary to a target RNA and wherein the methods further comprisedetecting the levels of target RNA.

In certain embodiments, an in vitro method of inhibiting gene expressionis provided comprising contacting one or more cells or a tissue with anoligomeric compound or a double stranded composition as provided herein.

In certain embodiments, an oligomeric or a double stranded compositionas provided herein is used in an in vivo method of inhibiting geneexpression, the method comprising contacting one or more cells, a tissueor an animal with an oligomeric or a double stranded composition asprovided herein.

In certain embodiments, an oligomeric or a double stranded compositionas provided herein; is used in medical therapy.

In certain embodiments, the invention provides pharmaceuticalcompositions comprising an oligomeric compound and a pharmaceuticallyacceptable diluent or carrier. In certain embodiments, thepharmaceutically acceptable diluent or carrier is pharmaceutical gradesterile saline.

In certain embodiments, the invention provides methods comprisingcontacting a cell with an oligomeric compound described herein. Incertain embodiments, such methods comprise detecting antisense activity.In certain embodiments, the detecting antisense activity comprisesdetecting a phenotypic change in the cell. In certain embodiments, thedetecting antisense activity comprises detecting a change in the amountof target nucleic acid in the cell. In certain embodiments, thedetecting antisense activity comprises detecting a change in the amountof a target protein. In certain embodiments, the cell is in vitro. Incertain embodiments, the cell is in an animal. In certain embodiments,animal is a mammal. In certain embodiments, the mammal is a human.

In certain embodiments, the invention provides methods of modulating atarget mRNA in a cell comprising contacting the cell with an oligomericcompound of the invention and thereby modulating the mRNA in a cell. Incertain embodiments, such methods comprise detecting a phenotypic changein the cell. In certain embodiments, methods comprise detecting adecrease in mRNA levels in the cell. In certain embodiments, methodscomprise detecting a change in the amount of a target protein. Incertain embodiments, the cell is in vitro. In certain embodiments, thecell is in an animal. In certain embodiments, the animal is a mammal. Incertain embodiments, the mammal is a human.

In certain embodiments, the invention provides methods of administeringto an animal a pharmaceutical composition of the invention. In certainembodiments, the animal is a mammal. In certain embodiments, the mammalis a human. In certain embodiments, the methods comprise detectingantisense activity in the animal. In certain embodiments, the methodscomprise detecting a change in the amount of target nucleic acid in theanimal. In certain embodiments, the methods comprise detecting a changein the amount of a target protein in the animal. In certain embodiments,the methods comprise detecting a phenotypic change in the animal. Incertain embodiments, the phenotypic change is a change in the amount orquality of a biological marker of activity.

In certain embodiments, the invention provides use of an oligomericcompound of the invention for the manufacture of a medicament for thetreatment of a disease characterized by undesired gene expression.

In certain embodiments, the invention provides use of an oligomericcompound of the invention for the manufacture of a medicament fortreating a disease by inhibiting gene expression.

In certain embodiments, the invention provides methods of comprisingdetecting antisense activity wherein the antisense activity is microRNAmimic activity. In certain embodiments, the detecting microRNA mimicactivity comprises detecting a change in the amount of a target nucleicacid in a cell. In certain embodiments, the detecting microRNA mimicactivity comprises detecting a change in the amount of a target proteinin cell.

DETAILED DESCRIPTION OF THE INVENTION

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,18th edition, 1990; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosureherein are incorporated by reference in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “nucleoside” refers to a compound comprising aheterocyclic base moiety and a sugar moiety. Nucleosides include, butare not limited to, naturally occurring nucleosides (as found in DNA andRNA), abasic nucleosides, modified nucleosides, and nucleosides havingmimetic bases and/or sugar groups. Nucleosides may be modified with anyof a variety of substituents. Nucleosides may include a phosphatemoiety.

As used herein, “sugar moiety” means a natural or modified sugar ring orsugar surrogate.

As used herein the term “sugar surrogate” refers to a structure that iscapable of replacing the furanose ring of a naturally occurringnucleoside. In certain embodiments, sugar surrogates are non-furanose(or 4′-substituted furanose) rings or ring systems or open systems. Suchstructures include simple changes relative to the natural furanose ring,such as a six membered ring or may be more complicated as is the casewith the non-ring system used in peptide nucleic acid. Sugar surrogatesincludes without limitation morpholinos, cyclohexenyls andcyclohexitols. In most nucleosides having a sugar surrogate group theheterocyclic base moiety is generally maintained to permithybridization.

As used herein, “nucleotide” refers to a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes “linkednucleotides.”

As used herein, “nucleobase” refers to the heterocyclic base portion ofa nucleoside. Nucleobases may be naturally occurring or may be modified.In certain embodiments, a nucleobase may comprise any atom or group ofatoms capable of hydrogen bonding to a base of another nucleic acid.

As used herein, “modified nucleoside” refers to a nucleoside comprisingat least one modification compared to naturally occurring RNA or DNAnucleosides. Such modification may be at the sugar moiety and/or at thenucleobases.

As used herein, “bicyclic nucleoside” or “BNA” refers to a nucleosidehaving a sugar moiety comprising a sugar-ring (including, but notlimited to, furanose) comprising a bridge connecting two carbon atoms ofthe sugar ring to form a second ring. In certain embodiments, the bridgeconnects the 4′ carbon to the 2′ carbon of a 5-membered sugar ring.

As used herein, “4′-2′ bicyclic nucleoside” refers to a bicyclicnucleoside comprising a furanose ring comprising a bridge connecting twocarbon atoms of the furanose ring connects the 2′ carbon atom and the 4′carbon atom of the sugar ring.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′ substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

As used herein “oligonucleoside” refers to an oligonucleotide in whichnone of the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” refers to an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein “internucleoside linkage” refers to a covalent linkagebetween adjacent nucleosides.

As used herein “naturally occurring internucleoside linkage” refers to a3′ to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” refers to anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” refers to a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound is an oligonucleotide. In certain embodiments, anoligomeric compound comprises one or more conjugate groups and/orterminal groups.

As used herein, unless otherwise indicated or modified, the term“double-stranded” or refers to two separate oligomeric compounds thatare hybridized to one another. Such double stranded compounds may haveone or more or non-hybridizing nucleosides at one or both ends of one orboth strands (overhangs) and/or one or more internal non-hybridizingnucleosides (mismatches) provided there is sufficient complementarity tomaintain hybridization under physiologically relevant conditions.

As used herein, the term “self-complementary” or “hair-pin” refers to asingle oligomeric compound that comprises a duplex region formed by theoligomeric compound hybridizing to itself.

As used herein, the term “single-stranded” refers to an oligomericcompound that is not hybridized to its complement and that does not havesufficient self-complementarity to form a hair-pin structure underphysiologically relevant conditions. A single-stranded compound may becapable of binding to its complement to become a double-stranded orpartially double-stranded compound.

As used herein, “terminal group” refers to one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more additionalnucleosides.

As used herein, “conjugate” refers to an atom or group of atoms bound toan oligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmakodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and clearance. Conjugate groups are routinely used in thechemical arts and are linked directly or via an optional linking moietyor linking group to the parent compound such as an oligomeric compound.In certain embodiments, conjugate groups includes without limitation,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,rhodamines, coumarins and dyes. In certain embodiments, conjugates areterminal groups. In certain embodiments, conjugates are attached to a 3′or 5′ terminal nucleoside or to an internal nucleosides of anoligonucleotide.

As used herein, “conjugate linking group” refers to any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound. Linking groups or bifunctional linking moieties such as thoseknown in the art are amenable to the present invention.

As used herein, “antisense compound” refers to an oligomeric compound,at least a portion of which is at least partially complementary to atarget nucleic acid to which it hybridizes. In certain embodiments, anantisense compound modulates (increases or decreases) expression oramount of a target nucleic acid. In certain embodiments, an antisensecompound alters splicing of a target pre-mRNA resulting in a differentsplice variant. In certain embodiments, an antisense compound modulatesexpression of one or more different target proteins. Antisensemechanisms contemplated herein include, but are not limited to an RNaseH mechanism, RNAi mechanisms, splicing modulation, translational arrest,altering RNA processing, inhibiting microRNA function, or mimickingmicroRNA function.

As used herein, “expression” refers to the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, splicing, post-transcriptional modification, andtranslation.

As used herein, “RNAi” refers to a mechanism by which certain antisensecompounds effect expression or amount of a target nucleic acid. RNAimechanisms involve the RISC pathway.

As used herein, “RNAi compound” refers to an oligomeric compound thatacts, at least in part, through an RNAi mechanism to modulate a targetnucleic acid and/or protein encoded by a target nucleic acid. RNAicompounds include, but are not limited to double-stranded shortinterfering RNA (siRNA), single-stranded RNA (ssRNA), and microRNA,including microRNA mimics.

As used herein, “antisense oligonucleotide” refers to an antisensecompound that is an oligonucleotide.

As used herein, “antisense activity” refers to any detectable and/ormeasurable activity attributable to the hybridization of an antisensecompound to its target nucleic acid. In certain embodiments, suchactivity may be an increase or decrease in an amount of a nucleic acidor protein. In certain embodiments, such activity may be a change in theratio of splice variants of a nucleic acid or protein. Detection and/ormeasuring of antisense activity may be direct or indirect. For example,in certain embodiments, antisense activity is assessed by detectingand/or measuring the amount of target protein or the relative amounts ofsplice variants of a target protein. In certain embodiments, antisenseactivity is assessed by detecting and/or measuring the amount of targetnucleic acids and/or cleaved target nucleic acids and/or alternativelyspliced target nucleic acids. In certain embodiments, antisense activityis assessed by observing a phenotypic change in a cell or animal.

As used herein “detecting” or “measuring” in connection with anactivity, response, or effect indicate that a test for detecting ormeasuring such activity, response, or effect is performed. Suchdetection and/or measuring may include values of zero. Thus, if a testfor detection or measuring results in a finding of no activity (activityof zero), the step of detecting or measuring the activity hasnevertheless been performed. For example, in certain embodiments, thepresent invention provides methods that comprise steps of detectingantisense activity, detecting toxicity, and/or measuring a marker oftoxicity. Any such step may include values of zero.

As used herein, “target nucleic acid” refers to any nucleic acidmolecule the expression, amount, or activity of which is capable ofbeing modulated by an antisense compound. In certain embodiments, thetarget nucleic acid is DNA or RNA. In certain embodiments, the targetRNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA,mature microRNA, promoter-directed RNA, or natural antisensetranscripts. For example, the target nucleic acid can be a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. In certain embodiments, target nucleic acid is aviral or bacterial-nucleic acid.

As used herein, “target mRNA” refers to a pre-selected RNA molecule thatencodes a protein.

As used herein, “target pre-mRNA” refers to a pre-selected RNAtranscript that has not been fully processed into mRNA. Notably, pre-RNAincludes one or more intron.

As used herein, “target microRNA” refers to a pre-selected non-codingRNA molecule about 18-30 nucleobases in length that modulates expressionof one or more proteins or to a precursor of such a non-coding molecule.

As used herein, “target pdRNA” refers to refers to a pre-selected RNAmolecule that interacts with one or more promoter to modulatetranscription.

As used herein, “microRNA” refers to a naturally occurring, small,non-coding RNA that represses gene expression at the level oftranslation. In certain embodiments, a microRNA represses geneexpression by binding to a target site within a 3′ untranslated regionof a target nucleic acid. In certain embodiments, a microRNA has anucleobase sequence as set forth in miRBase, a database of publishedmicroRNA sequences found at http://microrna.sanger.ac.uk/sequences/. Incertain embodiments, a microRNA has a nucleobase sequence as set forthin miRBase version 10.1 released December 2007, which is hereinincorporated by reference in its entirety. In certain embodiments, amicroRNA has a nucleobase sequence as set forth in miRBase version 12.0released September 2008, which is herein incorporated by reference inits entirety.

As used herein, “microRNA mimic” refers to an oligomeric compound havinga sequence that is at least partially identical to that of a microRNA.In certain embodiments, a microRNA mimic comprises the microRNA seedregion of a microRNA. In certain embodiments, a microRNA mimic modulatestranslation of more than one target nucleic acids.

As used herein, “seed region” refers to a region at or near the 5′ endof an antisense compound having a nucleobase sequence that is import fortarget nucleic acid recognition by the antisense compound. In certainembodiments, a seed region comprises nucleobases 2-8 of an antisensecompound. In certain embodiments, a seed region comprises nucleobases2-7 of an antisense compound. In certain embodiments, a seed regioncomprises nucleobases 1-7 of an antisense compound. In certainembodiments, a seed region comprises nucleobases 1-6 of an antisensecompound. In certain embodiments, a seed region comprises nucleobases1-8 of an antisense compound.

As used herein, “microRNA seed region” refers to a seed region of amicroRNA or microRNA mimic. In certain embodiments, a microRNA seedregion comprises nucleobases 2-8 of a microRNA or microRNA mimic. Incertain embodiments, a microRNA seed region comprises nucleobases 2-7 ofa microRNA or microRNA mimic. In certain embodiments, a microRNA seedregion comprises nucleobases 1-7 of a microRNA or microRNA mimic. Incertain embodiments, a microRNA seed region comprises nucleobases 1-6 ofa microRNA or microRNA mimic. In certain embodiments, a microRNA seedregion comprises nucleobases 1-8 of a microRNA or microRNA mimic.

As used herein, “seed match segment” refers to a portion of a targetnucleic acid having nucleobase complementarity to a seed region. Incertain embodiments, a seed match segment has nucleobase complementarityto nucleobases 2-8 of an siRNA, ssRNA, natural microRNA or microRNAmimic. In certain embodiments, a seed match segment has nucleobasecomplementarity to nucleobases 2-7 of an siRNA, ssRNA, microRNA ormicroRNA mimic. In certain embodiments, a seed match segment hasnucleobase complementarity to nucleobases 1-6 of an siRNA, ssRNA,microRNA or microRNA mimic. In certain embodiments, a seed match segmenthas nucleobase complementarity to nucleobases 1-7 of an siRNA, ssRNA,microRNA or microRNA mimic. In certain embodiments, a seed match segmenthas nucleobase complementarity to nucleobases 1-8 of an siRNA, ssRNA,microRNA or microRNA mimic.

As used herein, “seed match target nucleic acid” refers to a targetnucleic acid comprising a seed match segment.

As used herein, “microRNA family” refers to a group of microRNAs thatshare a microRNA seed sequence. In certain embodiments, microRNA familymembers regulate a common set of target nucleic acids. In certainembodiments, the shared microRNA seed sequence is found at the samenucleobase positions in each member of a microRNA family. In certainembodiments, the shared microRNA seed sequence is not found at the samenucleobase positions in each member of a microRNA family. For example, amicroRNA seed sequence found at nucleobases 1-7 of one member of amicroRNA family may be found at nucleobases 2-8 of another member of amicroRNA family.

As used herein, “target non-coding RNA” refers to a pre-selected RNAmolecule that is not translated to generate a protein. Certainnon-coding RNA are involved in regulation of expression.

As used herein, “target viral nucleic acid” refers to a pre-selectednucleic acid (RNA or DNA) associated with a virus. Such viral nucleicacid includes nucleic acids that constitute the viral genome, as well astranscripts (including reverse-transcripts and RNA transcribed from RNA)of those nucleic acids, whether or not produced by the host cellularmachinery. In certain instances, viral nucleic acids also include hostnucleic acids that are recruited by a virus upon viral infection.

As used herein, “targeting” or “targeted to” refers to the associationof an antisense compound to a particular target nucleic acid molecule ora particular region of nucleotides within a target nucleic acidmolecule. An antisense compound targets a target nucleic acid if it issufficiently complementary to the target nucleic acid to allowhybridization under physiological conditions.

As used herein, “target protein” refers to a protein, the expression ofwhich is modulated by an antisense compound. In certain embodiments, atarget protein is encoded by a target nucleic acid. In certainembodiments, expression of a target protein is otherwise influenced by atarget nucleic acid.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases refers to a nucleobase that is capable ofbase pairing with another nucleobase. For example, in DNA, adenine (A)is complementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase refers to a nucleobase of an antisense compound that iscapable of base pairing with a nucleobase of its target nucleic acid.For example, if a nucleobase at a certain position of an antisensecompound is capable of hydrogen bonding with a nucleobase at a certainposition of a target nucleic acid, then the position of hydrogen bondingbetween the oligonucleotide and the target nucleic acid is considered tobe complementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases refersto a pair of nucleobases that do not form hydrogen bonds with oneanother or otherwise support hybridization.

As used herein, “complementary” in reference to linked nucleosides,oligonucleotides, or nucleic acids, refers to the capacity of anoligomeric compound to hybridize to another oligomeric compound ornucleic acid through nucleobase complementarity. In certain embodiments,an antisense compound and its target are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatmay comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget). Preferably the antisense compounds contain no more than about15%, more preferably not more than about 10%, most preferably not morethan 5% or no mismatches. The remaining nucleotides are nucleobasecomplementary or otherwise do not disrupt hybridization (e.g., universalbases). One of ordinary skill in the art would recognize the compoundsprovided herein are at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%complementary to a target nucleic acid.

As used herein, “hybridization” refers to the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,the natural base adenine is nucleobase complementary to the naturalnucleobases thymidine and uracil which pair through the formation ofhydrogen bonds. The natural base guanine is nucleobase complementary tothe natural bases cytosine and 5-methyl cytosine. Hybridization canoccur under varying circumstances.

As used herein, “specifically hybridizes” refers to the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an antisense oligonucleotide specifically hybridizes tomore than one target site.

As used herein, “modulation” refers to a perturbation of amount orquality of a function or activity when compared to the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As a further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing, resulting in a change in the amount of a particularsplice-variant present compared to conditions that were not perturbed.As a further example, modulation includes perturbing translation of aprotein.

As used herein, “motif” refers to a pattern of modifications in anoligomeric compound or a region thereof. Motifs may be defined bymodifications at certain nucleosides and/or at certain linking groups ofan oligomeric compound.

As used herein, “nucleoside motif” refers to a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “linkage motif” refers to a pattern of linkagemodifications in an oligomeric compound or region thereof. Thenucleosides of such an oligomeric compound may be modified orunmodified. Unless otherwise indicated, motifs herein describing onlylinkages are intended to be linkage motifs. Thus, in such instances, thenucleosides are not limited.

As used herein, “different modifications” or “differently modified”refer to modifications relative to naturally occurring molecules thatare different from one another, including absence of modifications.Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are“differently modified,” even though the DNA nucleoside is unmodified.Likewise, DNA and RNA are “differently modified,” even though both arenaturally-occurring unmodified nucleosides. Nucleosides that are thesame but for comprising different nucleobases are not differentlymodified, unless otherwise indicated. For example, a nucleosidecomprising a 2′-OMe modified sugar and an adenine nucleobase and anucleoside comprising a 2′-OMe modified sugar and a thymine nucleobaseare not differently modified.

As used herein, “the same modifications” refer to modifications relativeto naturally occurring molecules that are the same as one another,including absence of modifications. Thus, for example, two unmodifiedDNA nucleoside have “the same modification,” even though the DNAnucleoside is unmodified.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” refers to the modification of a nucleoside andincludes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “separate regions” refers to a portion of an oligomericcompound wherein the nucleosides and internucleoside linkages within theregion all comprise the same modifications; and the nucleosides and/orthe internucleoside linkages of any neighboring portions include atleast one different modification.

As used herein, “alternating motif” refers to an oligomeric compound ora portion thereof, having at least four separate regions of modifiednucleosides in a pattern (AB)_(n)A_(m) where A represents a region ofnucleosides having a first type of modification; B represent a region ofnucleosides having a different type of modification; n is 2-15; and m is0 or 1. Thus, in certain embodiments, alternating motifs include 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or morealternating regions. In certain embodiments, each A region and each Bregion independently comprises 1-4 nucleosides.

As used herein, “fully modified” refers to an oligomeric compound orportion thereon wherein each nucleoside is a modified nucleoside. Themodifications of the nucleosides of a fully modified oligomeric compoundmay all be the same or one or more may be different from one another.

As used herein, “uniform modified” or “uniformly modified” refer tooligomeric compounds or portions thereof that comprise the samemodifications. The nucleosides of a region of uniformly modifiednucleosides all comprise the same modification.

As used herein the term “gapmer” or “gapped oligomeric compound” refersto an oligomeric compound having two external regions or wings and aninternal region or gap. The three regions form a contiguous sequence ofmonomer subunits with the sugar groups of the external regions beingdifferent than the sugar groups of the internal region and wherein thesugar group of each monomer subunit within a particular region isessentially the same.

As used herein, “pharmaceutically acceptable carrier or diluent” refersto any substance suitable for use in administering to an animal. Incertain embodiments, a pharmaceutically acceptable carrier or diluent issterile saline. In certain embodiments, such sterile saline ispharmaceutical grade saline.

The terms “substituent” and “substituent group,” as used herein, aremeant to include groups that are typically added to other groups orparent compounds to enhance desired properties or provide other desiredeffects. Substituent groups can be protected or unprotected and can beadded to one available site or to many available sites in a parentcompound. Substituent groups may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Substituent groups amenable herein include without limitation, halogen,hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl(—C(O)O—R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substitutedoxy (—O—R_(aa)), aryl, aralkyl, heterocyclic radical, heteroaryl,heteroarylalkyl, amino (—N(R_(bb))(R_(cc))), imino(═NR_(bb)), amido(—C(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro(—NO₂), cyano (—CN), carbamido ( OC(O)N(R_(bb))(R_(cc)) or—N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)N(R_(bb))(R_(cc))),thioureido (—N(R_(bb))C(S)N(R_(bb))—(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S—(O)₂R_(bb)).Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, anoptionally linked chemical functional group or a further substituentgroup with a preferred list including without limitation, H, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl. Selected substituentswithin the compounds described herein are present to a recursive degree.

In this context, “recursive substituent” means that a substituent mayrecite another instance of itself. Because of the recursive nature ofsuch substituents, theoretically, a large number may be present in anygiven claim. One of ordinary skill in the art of medicinal chemistry andorganic chemistry understands that the total number of such substituentsis reasonably limited by the desired properties of the compoundintended. Such properties include, by way of example and not limitation,physical properties such as molecular weight, solubility or log P,application properties such as activity against the intended target andpractical properties such as ease of synthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in a claim of the invention, the total numberwill be determined as set forth above.

The terms “stable compound” and “stable structure” as used herein aremeant to indicate a compound that is sufficiently robust to surviveisolation to a useful degree of purity from a reaction mixture, andformulation into an efficacious therapeutic agent, Only stable compoundsare contemplated herein.

The term “alkyl,” as used herein, refers to a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred. The term “lower alkyl” asused herein includes from 1 to about 6 carbon atoms. Alkyl groups asused herein may optionally include one or more further substituentgroups.

The term “alkenyl,” as used herein, refers to a straight or branchedhydrocarbon chain radical containing up to twenty four carbon atoms andhaving at least one carbon-carbon double bond. Examples of alkenylgroups include without limitation, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like.Alkenyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkenyl groups as used herein may optionallyinclude one or more further substituent groups.

The term “alkynyl,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms and havingat least one carbon-carbon triple bond. Examples of alkynyl groupsinclude, without limitation, ethynyl, 1-propynyl, 1-butynyl, and thelike. Alkynyl groups typically include from 2 to about 24 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred. Alkynyl groups as used herein mayoptionally include one or more further substituent groups.

The term “acyl,” as used herein, refers to a radical formed by removalof a hydroxyl group from an organic acid and has the general Formula—C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examplesinclude aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls,aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

The term “alicyclic” refers to a cyclic ring system wherein the ring isaliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

The term “aliphatic,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms with from 1to about 6 carbon atoms being more preferred. The straight or branchedchain of an aliphatic group may be interrupted with one or moreheteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Suchaliphatic groups interrupted by heteroatoms include without limitation,polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines.Aliphatic groups as used herein may optionally include furthersubstituent groups.

The term “alkoxy,” as used herein, refers to a radical formed between analkyl group and an oxygen atom wherein the oxygen atom is used to attachthe alkoxy group to a parent molecule. Examples of alkoxy groups includewithout limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.Alkoxy groups as used herein may optionally include further substituentgroups.

The term “aminoalkyl” as used herein, refers to an amino substitutedC₁-C₁₂ alkyl radical. The alkyl portion of the radical forms a covalentbond with a parent molecule. The amino group can be located at anyposition and the aminoalkyl group can be substituted with a furthersubstituent group at the alkyl and/or amino portions.

The terms “aralkyl” and “arylalkyl,” as used herein, refer to anaromatic group that is covalently linked to a C₁-C₁₂ alkyl radical. Thealkyl radical portion of the resulting aralkyl (or arylalkyl) groupforms a covalent bond with a parent molecule. Examples include withoutlimitation, benzyl, phenethyl and the like. Aralkyl groups as usedherein may optionally include further substituent groups attached to thealkyl, the aryl or both groups that form the radical group.

The terms “aryl” and “aromatic,” as used herein, refer to a mono- orpolycyclic carbocyclic ring system radicals having one or more aromaticrings. Examples of aryl groups include without limitation, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferredaryl ring systems have from about 5 to about 20 carbon atoms in one ormore rings. Aryl groups as used herein may optionally include furthersubstituent groups.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “heteroaryl,” and “heteroaromatic,” as used herein, refer to aradical comprising a mono- or poly-cyclic aromatic ring, ring system orfused ring system wherein at least one of the rings is aromatic andincludes one or more heteroatoms. Heteroaryl is also meant to includefused ring systems including systems where one or more of the fusedrings contain no heteroatoms. Heteroaryl groups typically include onering atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include without limitation, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like.Heteroaryl radicals can be attached to a parent molecule directly orthrough a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstituent groups.

The term “heteroarylalkyl,” as used herein, refers to a heteroaryl groupas previously defined that further includes a covalently attached C₁-C₁₂alkyl radical. The alkyl radical portion of the resultingheteroarylalkyl group is capable of forming a covalent bond with aparent molecule. Examples include without limitation, pyridinylmethyl,pyrimidinylethyl, napthyridinylpropyl and the like. Heteroarylalkylgroups as used herein may optionally include further substituent groupson one or both of the heteroaryl or alkyl portions.

The term “heterocyclic radical” as used herein, refers to a radicalmono-, or poly-cyclic ring system that includes at least one heteroatomand is unsaturated, partially saturated or fully saturated, therebyincluding heteroaryl groups. Heterocyclic is also meant to include fusedring systems wherein one or more of the fused rings contain at least oneheteroatom and the other rings can contain one or more heteroatoms oroptionally contain no heteroatoms. A heterocyclic radical typicallyincludes at least one atom selected from sulfur, nitrogen or oxygen.Examples of heterocyclic radicals include, [1,3]dioxolanyl,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and the like. Heterocyclic groups as used herein mayoptionally include further substituent groups.

The term “hydrocarbyl” includes radical groups that comprise C, O and H.Included are straight, branched and cyclic groups having any degree ofsaturation. Such hydrocarbyl groups can include one or more heteroatomsselected from N, O and S and can be further mono or poly substitutedwith one or more substituent groups.

The term “mono or poly cyclic structure” as used herein includes allring systems selected from single or polycyclic radical ring systemswherein the rings are fused or linked and is meant to be inclusive ofsingle and mixed ring systems individually selected from aliphatic,alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic,heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and polycyclic structures can contain rings that each have the same level ofsaturation or each, independently, have varying degrees of saturationincluding fully saturated, partially saturated or fully unsaturated.Each ring can comprise ring atoms selected from C, N, O and S to giverise to heterocyclic rings as well as rings comprising only C ring atomswhich can be present in a mixed motif such as for example benzimidazolewherein one ring has only carbon ring atoms and the fused ring has twonitrogen atoms. The mono or poly cyclic structures can be furthersubstituted with substituent groups such as for example phthalimidewhich has two ═O groups attached to one of the rings. Mono or polycyclic structures can be attached to parent molecules using variousstrategies such as directly through a ring atom, through a substituentgroup or through a bifunctional linking moiety.

The term “oxo” refers to the group (═O).

Linking groups or bifunctional linking moieties such as those known inthe art are useful for attachment of chemical functional groups,conjugate groups, reporter groups and other groups to selective sites ina parent compound such as for example an oligomeric compound. Ingeneral, a bifunctional linking moiety comprises a hydrocarbyl moietyhaving two functional groups. One of the functional groups is selectedto bind to a parent molecule or compound of interest and the other isselected to bind to essentially any selected group such as a chemicalfunctional group or a conjugate group. In some embodiments, the linkercomprises a chain structure or a polymer of repeating units such asethylene glycols or amino acid units. Examples of functional groups thatare routinely used in bifunctional linking moieties include withoutlimitation, electrophiles for reacting with nucleophilic groups andnucleophiles for reacting with electrophilic groups. In someembodiments, bifunctional linking moieties include amino, hydroxyl,carboxylic acid, thiol, unsaturations (e.g., double or triple bonds),and the like. Some nonlimiting examples of bifunctional linking moietiesinclude 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include withoutlimitation, substituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀ alkynyl, wherein anonlimiting list of preferred substituent groups includes hydroxyl,amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy,halogen, alkyl, aryl, alkenyl and alkynyl.

The term “phosphate moiety” as used herein, refers to a terminalphosphate group that includes phosphates as well as modified phosphates.The phosphate moiety can be located at either terminus but is preferredat the 5′-terminal nucleoside. In one aspect, the terminal phosphate isunmodified having the formula —O—P(═O)(OH)OH. In another aspect, theterminal phosphate is modified such that one or more of the O and OHgroups are replaced with H, O, S, N(R) or alkyl where R is H, an aminoprotecting group or unsubstituted or substituted alkyl. In certainembodiments, the 5′ and or 3′ terminal group can comprise from 1 to 3phosphate moieties that are each, independently, unmodified (di ortri-phosphates) or modified.

As used herein, the term “phosphorus moiety” refers to a group havingthe formula:

wherein:

R_(a) and R_(c), are each, independently, OH, SH, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, aminoor substituted amino; and

R_(c) is O or S.

Phosphorus moieties included herein can be attached to a monomer, whichcan be used in the preparation of oligomeric compounds, wherein themonomer may be attached using O, S, NR_(d) or CR_(e)R_(f), wherein R_(d)includes without limitation H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl,C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or substituted acyl,and R_(e) and R_(f) each, independently, include without limitation H,halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy orsubstituted C₁-C₆ alkoxy. Such linked phosphorus moieties includewithout limitation, phosphates, modified phosphates, thiophosphates,modified thiophosphates, phosphonates, modified phosphonates,phosphoramidates and modified phosphoramidates.

The term “protecting group,” as used herein, refers to a labile chemicalmoiety which is known in the art to protect reactive groups includingwithout limitation, hydroxyl, amino and thiol groups, against undesiredreactions during synthetic procedures. Protecting groups are typicallyused selectively and/or orthogonally to protect sites during reactionsat other reactive sites and can then be removed to leave the unprotectedgroup as is or available for further reactions. Protecting groups asknown in the art are described generally in Greene's Protective Groupsin Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.

Groups can be selectively incorporated into oligomeric compounds asprovided herein as precursors. For example an amino group can be placedinto a compound as provided herein as an azido group that can bechemically converted to the amino group at a desired point in thesynthesis. Generally, groups are protected or present as precursors thatwill be inert to reactions that modify other areas of the parentmolecule for conversion into their final groups at an appropriate time.Further representative protecting or precursor groups are discussed inAgrawal et al., Protocols for Oligonucleotide Conjugates, Humana Press;New Jersey, 1994, 26, 1-72.

The term “orthogonally protected” refers to functional groups which areprotected with different classes of protecting groups, wherein eachclass of protecting group can be removed in any order and in thepresence of all other classes (see, Barany et al., J. Am. Chem. Soc.,1977, 99, 7363-7365; Barany et al., J. Am. Chem. Soc., 1980, 102,3084-3095). Orthogonal protection is widely used in for exampleautomated oligonucleotide synthesis. A functional group is deblocked inthe presence of one or more other protected functional groups which isnot affected by the deblocking procedure. This deblocked functionalgroup is reacted in some manner and at some point a further orthogonalprotecting group is removed under a different set of reactionconditions. This allows for selective chemistry to arrive at a desiredcompound or oligomeric compound.

Examples of hydroxyl protecting groups include without limitation,acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl(TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoro-acetyl,pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate,mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl,dimethoxytrityl (DMT), trimethoxytrityl,1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl(Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Wherein morecommonly used hydroxyl protecting groups include without limitation,benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,benzoyl, mesylate, tosylate, dimethoxytrityl (DMT),9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl(MOX).

Examples of protecting groups commonly used to protect phosphate andphosphorus hydroxyl groups include without limitation, methyl, ethyl,benzyl (Bn), phenyl, isopropyl, tert-butyl, allyl, cyclohexyl (cHex),4-methoxybenzyl, 4-chlorobenzyl, 4-nitrobenzyl, 4-acyloxybenzyl,2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, diphenylmethyl,4-methylthio-1-butyl, 2-(S-Acetylthio)ethyl (SATE), 2-cyanoethyl,2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl,2-(trimethylsilyl)ethyl (TSE), 2-(phenylthio)ethyl,2-(triphenylsilyl)ethyl, 2-(benzylsulfonyl)ethyl, 2,2,2-trichloroethyl,2,2,2-tribromoethyl, 2,3-dibromopropyl, 2,2,2-trifluoroethyl,thiophenyl, 2-chloro-4-tritylphenyl, 2-bromophenyl,2-[N-isopropyl-N-(4-methoxybenzoyl)amino]ethyl,4-(N-trifluoroacetylamino)butyl, 4-oxopentyl, 4-tritylaminophenyl,4-benzylaminophenyl and morpholino. Wherein more commonly used phosphateand phosphorus protecting groups include without limitation, methyl,ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, 4-methoxybenzyl,4-chlorobenzyl, 2-chlorophenyl and 2-cyanoethyl.

Examples of amino protecting groups include without limitation,carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyl-oxycarbonyl (Cbz); amide-protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; andimine- and cyclic imide-protecting groups, such as phthalimido anddithiasuccinoyl.

Examples of thiol protecting groups include without limitation,triphenylmethyl (trityl), benzyl (Bn), and the like.

In certain embodiments, oligomeric compounds as provided herein can beprepared having one or more optionally protected phosphorus containinginternucleoside linkages. Representative protecting groups forphosphorus containing internucleoside linkages such as phosphodiesterand phosphorothioate linkages include β-cyanoethyl, diphenylsilylethyl,δ-cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoroacetyl ethyl(META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See forexample U.S. Pat. Nos. 4,725,677 and Re. 34,069 (β-cyanoethyl); Beaucageet al., Tetrahedron, 1993, 49(10), 1925-1963; Beaucage et al.,Tetrahedron, 1993, 49(46), 10441-10488; Beaucage et al., Tetrahedron,1992, 48(12), 2223-2311.

In certain embodiments, compounds having reactive phosphorus groups areprovided that are useful for forming internucleoside linkages includingfor example phosphodiester and phosphorothioate internucleosidelinkages. Such reactive phosphorus groups are known in the art andcontain phosphorus atoms in P^(III) or P^(V) valence state including,but not limited to, phosphoramidite, H-phosphonate, phosphate triestersand phosphorus containing chiral auxiliaries. In certain embodiments,reactive phosphorus groups are selected from diisopropylcyanoethoxyphosphoramidite (—O*—P[N[(CH(CH₃)₂]₂]O(CH₂)₂CN) and H-phosphonate(—O*—P(═O)(H)OH), wherein the O* is provided from the Markush group forthe monomer. A preferred synthetic solid phase synthesis utilizesphosphoramidites (P^(III) chemistry) as reactive phosphites. Theintermediate phosphite compounds are subsequently oxidized to thephosphate or thiophosphate (P^(V) chemistry) using known methods toyield, phosphodiester or phosphorothioate internucleoside linkages.Additional reactive phosphates and phosphites are disclosed inTetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48,2223-2311).

Certain Monomeric Compounds

In certain embodiments, the invention provides modified 5′ diphosphatenucleosides having Formula I:

wherein:

Bx is a heterocyclic base moiety;

each Pg is a hydroxyl protecting group;

M₁ is H, OH or OR₁;

M₂ is OH, OR₁ or N(R₁)(R₂);

each R₁ and R₂ is, independently, alkyl or substituted alkyl;

r is 0 or 1;

A is O, S, CR₃R₄ or N(R₅);

R₃ and R₄ are each, independently H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

R₅ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or a protecting group;

Q₁ and Q₂ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₆)(R₇)]_(n)—[(C═O)_(m)—X]_(j)—Z;

each R₆ and R₇ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from H, halogen, OJ₁,N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂), C(=L)N(J₁)(J₂),

C(=L)N(H)—(CH₂)₂N(J₁)(J₂) or a mono or poly cyclic ring system;

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

when Q₁ and Q₂ are each H and G is H or OH then A is other than O.

In certain embodiments, the invention provides modified 5′ diphosphatenucleosides having Formula I and further having the configuration ofFormula Ia:

Certain Oligomeric Compounds

In certain embodiments, the invention provides oligomeric compoundscomprising a modified 5′ diphosphate nucleoside having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T₁ is an internucleoside linking group linking the compound of FormulaII to the remainder of the oligomeric compound;

each M₃ is, independently, H or a hydroxyl protecting group;

A is O, S, CR₃R₄ or N(R₅);

R₃ and R₄ are each, independently H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

R₅ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or a protecting group;

Q₁ and Q₂ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₆)(R₆)(R₇)]_(n)—[(C═O)_(m)—X]_(j)—Z;

each R₆ and R₇ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from H, halogen, OJ₁,N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂), C(=L)N(J₁)(J₂),C(=L)N(H)—(CH₂)₂N(J₁)(J₂) or a mono or poly cyclic ring system;

L is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

when Q₁ and Q₂ are each H and G is H or OH then A is other than O.

The oligomeric compounds provided herein may also include one or more 5′or 3′ terminal groups.

In certain embodiments, oligomeric compounds are provided comprising amodified 5′ diphosphate nucleoside having Formula Ha:

In certain embodiments, oligomeric compounds comprise a nucleoside ofFormula II or IIa. In certain such embodiments, the nucleoside ofFormula II or IIa is at the 5′-terminus. In certain such embodiments,the remainder of the oligomeric compound comprises one or moremodifications. Such modifications may include modified sugar moieties,modified nucleobases and/or modified internucleoside linkages. Certainsuch modifications which may be incorporated in an oligomeric compoundcomprising a nucleoside of Formula II or IIa is at the 5′-terminus areknown in the art.

Certain Modified Sugar Moieties

Oligomeric compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprise achemically modified ribofuranose ring moiety. Examples of chemicallymodified ribofuranose rings include, without limitation, addition ofsubstitutent groups (including 5′ and/or 2′ substituent groups; bridgingof two ring atoms to form bicyclic nucleic acids (BNA); replacement ofthe ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R=H, C₁-C₁₂alkyl or a protecting group); and combinations thereof. Examples ofchemically modified sugars include, 2′-F-5′-methyl substitutednucleoside (see, PCT International Application WO 2008/101157, publishedon Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides),replacement of the ribosyl ring oxygen atom with S with furthersubstitution at the 2′-position (see, published U.S. Patent ApplicationUS2005/0130923, published on Jun. 16, 2005), or, alternatively,5′-substitution of a BNA (see, PCT International Application WO2007/134181, published on Nov. 22, 2007, wherein LNA is substitutedwith, for example, a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include, withoutlimitation, nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, and 2′-O(CH₂)₂OCH₃ substituent groups. The substituent atthe 2′ position can also be selected from allyl, amino, azido, thio,O-allyl, O—C₁-C₁₀ alkyl, OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(Rm)(Rn), andO—CH₂—C(═O)—N(Rm)(Rn), where each R^(m) and R^(n) is, independently, Hor substituted or unsubstituted C₁-C₁₀ alkyl.

In certain embodiments, oligomeric compounds of the present inventioninclude one or mre bicyclic nucleoside. In certain such embodimetns, thebicyclic ncleoside comprises a bridge between the 4′ and the 2′ ribosylring atoms. In certain embodiments, oligomeric compounds provided hereininclude one or more bicyclic nucleosides wherein the bridge comprises a4′ to 2′ bicyclic nucleoside. Examples of such 4′ to 2′ bicyclicnucleosides, include, but are not limited to, one of the formulae:4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ and 4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see, U.S.Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ andanalogs thereof, (see, published International ApplicationWO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, published PCT International Application WO2008/150729,published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. PatentApplication US2004/0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (seeChattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008). Also see, forexample: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; International applications WO 2004/106356, WO 1994/14226, WO2005/021570, and WO 2007/134181; U.S. Patent Publication Nos.US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser.Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564,61/086,231, 61/097,787, and 61/099,844; and PCT InternationalApplications Nos. PCT/US2008/064591, PCT/US2008/066154, andPCT/US2008/068922. Each of the foregoing bicyclic nucleosides can beprepared having one or more stereochemical sugar configurationsincluding for example α-L-ribofuranose and β-D-ribofuranose (see PCTinternational application PCT/DK98/00393, published on Mar. 25, 1999 asWO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ position of the pentofuranosyl sugar moietywherein such bridges independently comprises 1 or from 2 to 4 linkedgroups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂—J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is4′-CH₂-2′,4′-(CH₂)₂-2′,4′-(CH₂)₃-2′,4′-CH₂—O-2′,4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R)-2′,and 4′-CH₂—N(R)—O-2′-, wherein each R is, independently, H, a protectinggroup, or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F)Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to asconstrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H)methylene-amino (4′-CH2-N(R)-2′) BNA, (I) methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic (4′-(CH₂)₃-2′)BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protectinggroup, or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleoside having Formula I:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O—, or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently, H, a hydroxylprotecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium.

In certain embodiments, bicyclic nucleoside having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol, or substituted thio.

In certain embodiments, each of the substituted groups is,independently, mono or poly substituted with substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d),SJ_(c), N₃, OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(a),J_(d), and J_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆alkyl and X is O or NJ_(c).

In certain embodiments, bicyclic nucleoside having Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleoside having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl, orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleoside having Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

q_(a), q_(b), q_(e), and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl, orsubstituted C₁-C₁₂ alkyl. The synthesis and preparation of themethyleneoxy (4′-CH₂—O-2′) BNA monomers adenine, cytosine, guanine,5-methyl-cytosine, thymine, and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (see, e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630).BNAs and preparation thereof are also described in WO 98/39352 and WO99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA, methyleneoxy (4′-CH₂—O-2′)BNA, and 2′-thio-BNAs, have also been prepared (see, e.g., Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of lockednucleoside analogs comprising oligodeoxyribonucleotide duplexes assubstrates for nucleic acid polymerases has also been described (see,e.g., Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel comformationally restricted high-affinityoligonucleotide analog, has been described in the art (see, e.g., Singhet al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino-and 2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In certain embodiments, bicyclic nucleoside having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), O—C(═O)NJ_(j)J_(k),N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k), or N(H)C(═S)NJ_(j)J_(k);and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl, or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog, bridge 4′-CH═CH—CH₂-2′, have been described (see, e.g.,Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 andAlbaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis andpreparation of carbocyclic bicyclic nucleosides along with theiroligomerization and biochemical studies have also been described (see,e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, oligomeric compounds comprise one or moremodified tetrahydropyran nucleoside, which is a nucleoside having asix-membered tetrahydropyran in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified tetrahydropyran nucleosidesinclude, but are not limited to, what is referred to in the art ashexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleicacid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854),fluoro HNA (F—HNA), or those compounds having Formula X:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula X areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula X areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).Combinations of these modifications are also provided for herein withoutlimitation, such as 2′-F-5′-methyl substituted nucleosides (see PCTInternational Application WO 2008/101157 Published on Aug. 21, 2008 forother disclosed 5′,2′-bis substituted nucleosides) and replacement ofthe ribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). Such ring systems can undergo various additional substitutionsto enhance activity.

Methods for the preparations of modified sugars are well known to thoseskilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified, or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleotides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleotides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a cEt. In certain embodiments,the cEt modified nucleotides are arranged throughout the wings of agapmer motif.

Certain Modified Nucleobases

In certain embodiments, nucleosides of the present invention compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present invention comprise one or more modified nucleobases.

As used herein the terms, “unmodified nucleobase” and “naturallyoccurring nucleobase” include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic and natural nucleobasessuch as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases,size-expanded bases, and fluorinated bases as defined herein. Furthermodified nucleobases include tricyclic pyrimidines such as phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

The heterocyclic base moiety of each of the nucleosides can be modifiedwith one or more substituent groups to enhance one or more propertiessuch as affinity for a target strand or affect some other property in anadvantageous manner. Modified nucleobases include without limitation,universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds as provided herein. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (AntisenseResearch and Applications, Sanghvi, Y. S., Crooke, S. T. and Lebleu, B.,Eds., CRC Press, Boca Raton, 1993, 276-278).

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

Certain Internucleoside Linkages

In certain embodiments, the present invention provides oligomericcompounds comprising linked nucleosides. In such embodiments,nucleosides may be linked together using any internucleoside linkage.The two main classes of internucleoside linking groups are defined bythe presence or absence of a phosphorus atom. Representative phosphoruscontaining internucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Oligonucleotides having non-phosphorus internucleoside linking groupsmay be referred to as oligonucleosides. Modified linkages, compared tonatural phosphodiester linkages, can be used to alter, typicallyincrease, nuclease resistance of the oligomeric compound. In certainembodiments, internucleoside linkages having a chiral atom can beprepared a racemic mixture, as separate enantiomers. Representativechiral linkages include, but are not limited to, alkylphosphonates andphosphorothioates. Methods of preparation of phosphorous-containing andnon-phosphorous-containing internucleoside linkages are well known tothose skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), □ or □ such as for sugar anomers, or as(D) or (L) such as for amino acids et al. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

As used herein the phrase “neutral internucleoside linkage” is intendedto include internucleoside linkages that are non-ionic. Neutralinternucleoside linkages include without limitation, phosphotriesters,methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Certain Lengths

In certain embodiments, the present invention provides oligomericcompounds including oligonucleotides of any of a variety of ranges oflengths. In certain embodiments, the invention provides oligomericcompounds or oligonucleotides consisting of X to Y linked nucleosides,where X represents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≦Y. For example, in certainembodiments, the invention provides oligomeric compounds which compriseoligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linkednucleosides. In embodiments where the number of nucleosides of anoligomeric compound or oligonucleotide is limited, whether to a range orto a specific number, the oligomeric compound or oligonucleotide may,nonetheless further comprise additional other substituents. For example,an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotideshaving 31 nucleosides, but, unless otherwise indicated, such anoligonucleotide may further comprise, for example one or moreconjugates, terminal groups, or other substituents. In certainembodiments, terminal groups include, but are not limited to, terminalgroup nucleosides. In such embodiments, the terminal group nucleosidesare differently modified than the terminal nucleoside of theoligonucleotide, thus distinguishing such terminal group nucleosidesfrom the nucleosides of the oligonucleotide.

Certain Motifs

In certain embodiments, the present invention provides oligomericcompounds comprising one or more regions having a particular nucleosidemotif. In certain embodiments, the 5′-terminal nucleoside of a modifiedoligonucleotide of the present invention comprises a compound of FormulaII or IIa.

Gapped Motifs

In certain embodiments, the oligomeric compounds of the presentinvention comprise a gapmer region. In certain such embodiments, thesugar groups of the external regions are the same as one another(referred to herein as a symmetric gapmer). In certain embodiments, thesugar group used in the 5′-external region is different from the sugargroup used in the 3′-external region (referred to herein as anasymmetric gapmer). In certain embodiments, the external regions aresmall (each independently 1, 2, 3, 4 or about 5 monomer subunits) andthe monomer subunits comprise non-naturally occurring sugar groups withthe internal region comprising β-D-2′-deoxyribonucleosides. In certainembodiments, the external regions each, independently, comprise from 1to about 5 monomer subunits having non-naturally occurring sugar groupsand the internal region comprises from 6 to 18 unmodified nucleosides.The internal region or the gap generally comprisesβ-D-2′-deoxyribonucleosides but can comprise non-naturally occurringsugar groups. The heterocyclic base and internucleoside linkage isindependently variable at each position of a gapped oligomeric compound.The motif further optionally includes the use of one or more othergroups including but not limited to capping groups, conjugate groups andother 5′ or 3′-terminal groups.

In certain embodiments, the gapped oligomeric compounds comprise aninternal region of β-D-2′-deoxyribonucleosides with one of the externalregions comprising modified nucleosides. In certain embodiments, thegapped oligomeric compounds comprise an internal region ofβ-D-2′-deoxyribonucleosides with both of the external regions comprisingmodified nucleosides. In certain embodiments, gapped oligomericcompounds are provided herein wherein all of the monomer subunitscomprise non-naturally occurring sugar groups.

In certain embodiments, gapped oligomeric compounds are providedcomprising one or two modified nucleosides at the 5′-end, two or threemodified nucleosides at the 3′-end and an internal region of from 10 to16 β-D-2′-deoxyribonucleosides. In certain embodiments, gappedoligomeric compounds are provided comprising one modified nucleoside atthe 5′-end, two modified nucleosides at the 3′-end and an internalregion of from 10 to 16 β-D-2′-deoxyribonucleosides. In certainembodiments, gapped oligomeric compounds are provided comprising onemodified nucleosides at the 5′-end, two modified nucleosides at the3′-end and an internal region of from 10 to 14β-D-2′-deoxyribonucleosides.

In certain embodiments, gapped oligomeric compounds are provided thatare from about 10 to about 21 monomer subunits in length. In certainembodiments, gapped oligomeric compounds are provided that are fromabout 12 to about 16 monomer subunits in length. In certain embodiments,gapped oligomeric compounds are provided that are from about 12 to about14 monomer subunits in length.

Certain alternating regions

In certain embodiments, oligonucleotides of the present inventioncomprise one or more regions of alternating modifications. In certainembodiments, oligonucleotides comprise one or more regions ofalternating nucleoside modifications. In certain embodiments,oligonucleotides comprise one or more regions of alternating linkagemodifications. In certain embodiments, oligonucleotides comprise one ormore regions of alternating nucleoside and linkage modifications.

In certain embodiments, oligonucleotides of the present inventioncomprise one or more regions of alternating 2′-F modified nucleosidesand 2′-OMe modified nucleosides. In certain such embodiments, suchregions of alternating 2′F modified and 2′OMe modified nucleosides alsocomprise alternating linkages. In certain such embodiments, the linkagesat the 3′ end of the 2′-F modified nucleosides are phosphorothioatelinkages. In certain such embodiments, the linkages at the 3′ end of the2′OMe nucleosides are phosphodiester linkages. In certain embodiments,such alternating regions are:

(2′-F)—(PS)-(2′-OMe)-(PO)

In certain embodiments, oligomeric compounds comprise 2, 3, 4, 5, 6, 7,8, 9, 10, or 11 such alternating regions. Such regions may be contiguousor may be interrupted by differently modified nucleosides or linkages.

In certain embodiments, one or more alternating regions in analternating motif include more than a single nucleoside of a type. Forexample, oligomeric compounds of the present invention may include oneor more regions of any of the following nucleoside motifs:

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA; or

ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of asecond type. In certain embodiments, A and B are each selected from2′-F, 2′-OMe, BNA, DNA, and MOE.

In certain embodiments, A is DNA. In certain embodiments, B is4′-CH₂O-2′-BNA. In certain embodiments, A is DNA and B is4′-CH₂O-2′-BNA. In certain embodiments A is 4′-CH₂O-2′-BNA. In certainembodiments, B is DNA. In certain embodiments A is 4′-CH₂O-2′-BNA and Bis DNA. In certain embodiments, A is 2′-F. In certain embodiments, B is2′-OMe. In certain embodiments, A is 2′-F and B is 2′-OMe. In certainembodimentns, A is 2′-OMe. In certain embodiments, B is 2′-F. In certainembodiments, A is 2′-OMe and B is 2′-F. In certain embodiments, A is DNAand B is 2′-OMe. In certain embodiments, A is 2′-OMe and B is DNA.

In certain embodiments, oligomeric compounds having such an alternatingmotif also comprise a 5′ terminal nucleoside of formula II or IIa.

Two-Two-Three Motifs

In certain embodiments, oligonucleotides of the present inventioncomprise a region having a 2-2-3 motif. Such regions comprises thefollowing motif:

5′-(Formula II or IIa)-(E)_(w)-(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-(D)_(z)

wherein: A is a first type of modified nucleoside;

B, C, D, and E are nucleosides that are differently modified than A,however, B, C, D, and E may have the same or different modifications asone another;

w and z are from 0 to 15;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certainembodiments, B, C, D, and E are all 2′-F modified nucleosides. Incertain embodiments, A is a 2′-OMe modified nucleoside and B, C, D, andE are all 2′-F modified nucleosides.

In certain embodiments, the linkages of a 2-2-3 motif are all modifiedlinkages. In certain embodiments, the linkages are all phosphorothioatelinkages. In certain embodimentns, the linkages at the 3′-end of eachmodification of the first type are phosphodiester.

In certain embodiments, Z is 0. In such embodiments, the region of threenucleosides of the first type are at the 3′-end of the oligonucleotide.In certain embodiments, such region is at the 3′-end of the oligomericcompound, with no additional groups attached to the 3′ end of the regionof three nucleosides of the first type. In certain embodiments, anoligomeric compound comprising an oligonucleotide where Z is 0, maycomprise a terminal group attached to the 3′-terminal nucleoside. Suchterminal groups may include additional nucleosides. Such additionalnucleosides are typically non-hybridizing nucleosides.

In certain embodiments, Z is 1-3. In certain embodiments, Z is 2. Incertain embodiments, the nucleosides of Z are 2′-MOE nucleosides. Incertain embodiments, Z represents non-hybridizing nucleosides. To avoidconfusion, it is noted that such non-hybridizing nucleosides might alsobe described as a 3′-terminal group with Z=0.

Combinations of Motifs

It is to be understood, that certain of the above described motifs andmodifications may be combined. Since a motif may comprises only a fewnucleosides, a particular oligonucleotide may comprise two or moremotifs. By way of non-limiting example, in certain embodiments,oligomeric compounds may have nucleoside motifs as described in thetable below. In the table below, the term “None” indicates that aparticular feature is not present in the oligonucleotide. For example,“None” in the column labeled “5′ motif/modification” indicates that the5′ end of the oligonucleotide comprises the first nucleoside of thecentral motif.

5′ motif/modification Central Motif 3′-motif Compound of Formula II orIIa Alternating 2 MOE nucleosides Compound of Formula II or IIa 2-2-3motif 2 MOE nucleosides Compound of Formula II or IIa Uniform 2 MOEnucleosides Compound of Formula II or IIa Alternating 2 MOE nucleosidesCompound of Formula II or IIa Alternating 2 MOE A's Compound of FormulaII or IIa 2-2-3 motif 2 MOE A's Compound of Formula II or IIa Uniform 2MOE A's Compound of Formula II or IIa Alternating 2 MOE U's Compound ofFormula II or IIa 2-2-3 motif 2 MOE U's Compound of Formula II or IIaUniform 2 MOE U's Compound of Formula II or IIa Alternating 2 MOEnucleosides Compound of Formula II or IIa 2-2-3 motif 2 MOE nucleosidesCompound of Formula II or IIa Uniform 2 MOE nucleosidesOligomeric compounds having any of the various nucleoside motifsdescribed herein, may have any linkage motif. For example, theoligomeric compounds, including but not limited to those described inthe above table, may have a linkage motif selected from non-limiting thetable below:

5′ most linkage Central region 3′-region PS Alternating PO/PS 6 PS PSAlternating PO/PS 7 PS PS Alternating PO/PS 8 PS

As is apparent from the above, non-limiting tables, the lengths of theregions defined by a nucleoside motif and that of a linkage motif neednot be the same. For example, the 3′ region in the nucleoside motiftable above is 2 nucleosides, while the 3′-region of the linkage motiftable above is 6-8 nucleosides. Combining the tables results in anoligonucleotide having two 3′-terminal MOE nucleosides and six to eight3′-terminal phosphorothioate linkages (so some of the linkages in thecentral region of the nucleoside motif are phosphorothioate as well). Tofurther illustrate, and not to limit in any way, nucleoside motifs andsequence motifs are combined to show five non-limiting examples in thetable below. The first column of the table lists nucleosides andlinkages by position from N1 (the first nucleoside at the 5′-end) to N20(the 20^(th) position from the 5′-end). In certain embodiments,oligonucleotides of the present invention are longer than 20 nucleosides(the table is merely exemplary). Certain positions in the table recitethe nucleoside or linkage “none” indicating that the oligonucleotide hasno nucleoside at that position.

Pos A B C D E N1 Formula Formula Formula Formula Formula II or IIa II orIIa II or IIa II or IIa II or IIa L1 PS PS PS PS PO N2 2′-F 2′-F 2′-F2′-OMe MOE L2 PS PS PS PO PS N3 2′-OMe 2′-F 2′-F 2′-F 2′-F L3 PO PS PSPS PS N4 2′-F 2′-F 2′-F 2′-OMe 2′-F L4 PS PS PS PO PS N5 2′-OMe 2′-F2′-F 2′-F 2′-OMe L5 PO PS PS PS PO N6 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe L6PS PO PS PO PO N7 2′-OMe 2′-OMe 2′-F 2′-F 2′-OMe L7 PO PO PS PS PO N82′-F 2′-F 2′-F 2′-OMe 2′-F L8 PS PS PS PO PS N9 2′-OMe 2′-F 2′-F 2′-F2′-F L9 PO PS PS PS PS N10 2′-F 2′-OMe 2′-F 2′-OMe 2′-OMe L10 PS PO PSPO PO N11 2′-OMe 2′-OMe 2′-F 2′-F 2′OMe L11 PO PO PS PS PO N12 2′-F 2′-F2′-F 2′-F 2′-F L12 PS PS PS PO PS N13 2′-OMe 2′-F 2′-F 2′-F 2′-F L13 POPS PS PS PS N14 2′-F 2′-OMe 2′-F 2′-F 2′-F L14 PS PS PS PS PS N15 2′-OMe2′OMe 2′-F 2′-F 2′-MOE L15 PS PS PS PS PS N16 2′-F 2′OMe 2′-F 2′-F2′-MOE L16 PS PS PS PS PS N17 2′-OMe 2′-MOE U 2′-F 2′-F 2′-MOE L17 PS PSPS PS None N18 2′-F 2′-MOE U 2′-F 2′-OMe None L18 PS None PS PS None N192′-MOE U None 2′-MOE U 2′-MOE A None L19 PS None PS PS None N20 2′-MOE UNone 2′-MOE U 2′-MOE A NoneIn the above, non-limiting examples:

Column A represent an oligomeric compound consisting of 20 linkednucleosides, wherein the oligomeric compound comprises: a modified5′-terminal nucleoside of Formula II or IIa; a region of alternatingnucleosides; a region of alternating linkages; two 3′-terminal MOEnucleosides, each of which comprises a uracil base; and a region of sixphosphorothioate linkages at the 3′-end.

Column B represents an oligomeric compound consisting of 18 linkednucleosides, wherein the oligomeric compound comprises: a modified5′-terminal nucleoside of Formula II or IIa; a 2-2-3 motif wherein themodified nucleoside of the 2-2-3 motif are 2′O-Me and the remainingnucleosides are all 2′-F; two 3′-terminal MOE nucleosides, each of whichcomprises a uracil base; and a region of six phosphorothioate linkagesat the 3′-end.

Column C represents an oligomeric compound consisting of 20 linkednucleosides, wherein the oligomeric compound comprises: a modified5′-terminal nucleoside of Formula II or IIa; a region of uniformlymodified 2′-F nucleosides; two 3′-terminal MOE nucleosides, each ofwhich comprises a uracil base; and wherein each internucleoside linkageis a phosphorothioate linkage.

Column D represents an oligomeric compound consisting of 20 linkednucleosides, wherein the oligomeric compound comprises: a modified5′-terminal nucleoside of Formula II or IIa; a region of alternating2′-OMe/2′-F nucleosides; a region of uniform 2′F nucleosides; a regionof alternating phosphorothioate/phosphodiester linkages; two 3′-terminalMOE nucleosides, each of which comprises an adenine base; and a regionof six phosphorothioate linkages at the 3′-end.

Column E represents an oligomeric compound consisting of 17 linkednucleosides, wherein the oligomeric compound comprises: a modified5′-terminal nucleoside of Formula II or IIa; a 2-2-3 motif wherein themodified nucleoside of the 2-2-3 motif are 2′F and the remainingnucleosides are all 2′-OMe; three 3′-terminal MOE nucleosides.

The above examples are provided solely to illustrate how the describedmotifs may be used in combination and are not intended to limit theinvention to the particular combinations or the particular modificationsused in illustrating the combinations. Further, specific examplesherein, including, but not limited to those in the above table areintended to encompass more generic embodiments. For example, column A inthe above table exemplifies a region of alternating 2′-OMe and 2′-Fnucleosides. Thus, that same disclosure also exemplifies a region ofalternating different 2′-modifications. It also exemplifies a region ofalternating 2′-O-alkyl and 2′-halogen nucleosides. It also exemplifies aregion of alternating differently modified nucleosides. All of theexamples throughout this specification contemplate such genericinterpretation.

It is also noted that the lengths of oligomeric compounds, such as thoseexemplified in the above tables, can be easily manipulated bylengthening or shortening one or more of the described regions, withoutdisrupting the motif.

In certain embodiments, the intention provides oligomeric compoundswherein the 5′-terminal nucleoside (position 1) is a compound of FormulaII or IIa and the position 2 nucleoside comprises a 2′-modification. Incertain such embodiments, the 2′-modification of the position 2nucleoside is selected from halogen, alkyl, and substituted alkyl. Incertain embodiments, the 2′-modification of the position 2 nucleoside isselected from 2′-F and 2′-alkyl. In certain embodiments, the2′-modification of the position 2 nucleoside is 2′-F. In certainembodiments, the 2′-substitued of the position 2 nucleoside is anunmodified OH (as in naturally occurring RNA).

In certain embodiments, the position 3 nucleoside is a modifiednucleoside. In certain embodiments, the position 3 nucleoside is abicyclic nucleoside. In certain embodiments, the position 3 nucleosidecomprises a sugar surrogate. In certain such embodiments, the sugarsurrogate is a tetrahydropyran. In certain embodiments, the sugar of theposition 3 nucleoside is a F—HNA.

In certain embodiments, an antisense oligomeric compound comprises anoligonucleotide comprising 10 to 30 linked nucleosides wherein theoligonucleotide comprises:

a position 1 modified nucleoside of Formula II or IIa;

a position 2 nucleoside comprising a sugar moiety which is differentlymodified compared to the sugar moiety of the position 1 modifiednucleoside; and

from 1 to 4 3′-terminal group nucleosides each comprising a2′-modification; and

wherein at least the seven 3′-most internucleoside linkages arephosphorothioate linkages.

Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamics, pharmacokinetics, stability, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligomericcompound, such as an oligonucleotide. Conjugate groups includes withoutlimitation, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groupshave been described previously, for example: cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130.

Representative U.S. patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

In certain embodiments, conjugate groups are directly attached tooligonucleotides in oligomeric compounds. In certain embodiments,conjugate groups are attached to oligonucleotides by a conjugate linkinggroup. In certain such embodiments, conjugate linking groups, including,but not limited to, bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Conjugate linkinggroups are useful for attachment of conjugate groups, such as chemicalstabilizing groups, functional groups, reporter groups and other groupsto selective sites in a parent compound such as for example anoligomeric compound. In general a bifunctional linking moiety comprisesa hydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such aschemical functional group or a conjugate group. In some embodiments, theconjugate linker comprises a chain structure or an oligomer of repeatingunits such as ethylene glycol or amino acid units. Examples offunctional groups that are routinely used in a bifunctional linkingmoiety include, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties includepyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C1-C10 alkyl, substituted orunsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′ end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group. In certain embodiments, thepresent invention provides oligomeric compounds. In certain embodiments,oligomeric compounds comprise an oligonucleotide. In certainembodiments, an oligomeric compound comprises an oligonucleotide and oneor more conjugate and/or terminal groups. Such conjugate and/or terminalgroups may be added to oligonucleotides having any of the chemicalmotifs discussed above. Thus, for example, an oligomeric compoundcomprising an oligonucleotide having region of alternating nucleosidesmay comprise a terminal group.

Antisense Compounds

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. In such embodiments, the oligomeric compound iscomplementary to a target nucleic acid. In certain embodiments, a targetnucleic acid is an RNA. In certain embodiments, a target nucleic acid isa non-coding RNA. In certain embodiments, a target nucleic acid encodesa protein. In certain embodiments, a target nucleic acid is selectedfrom a mRNA, a pre-mRNA, a microRNA, a non-coding RNA, including smallnon-coding RNA, and a promoter-directed RNA. In certain embodiments,oligomeric compounds are at least partially complementary to more thanone target nucleic acid. For example, oligomeric compounds of thepresent invention may be microRNA mimics, which typically bind tomultiple targets.

Antisense mechanisms include any mechanism involving the hybridizationof an oligomeric compound with target nucleic acid, wherein thehybridization results in a biological effect. In certain embodiments,such hybridization results in either target nucleic acid degradation oroccupancy with concomitant inhibition or stimulation of the cellularmachinery involving, for example, translation, transcription, orsplicing of the target nucleic acid.

One type of antisense mechanism involving degradation of target RNA isRNase H mediated antisense. RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. It is known in the art thatsingle-stranded antisense compounds which are “DNA-like” elicit RNase Hactivity in mammalian cells. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof DNA-like oligonucleotide-mediated inhibition of gene expression.

Antisense mechanisms also include, without limitation RNAi mechanisms,which utilize the RISC pathway. Such RNAi mechanisms include, withoutlimitation siRNA, ssRNA and microRNA mechanisms. Such mechanism includecreation of a microRNA mimic and/or an anti-microRNA.

Antisense mechanisms also include, without limitation, mechanisms thathybridize or mimic non-coding RNA other than microRNA or mRNA. Suchnon-coding RNA includes, but is not limited to promoter-directed RNA andshort and long RNA that effects transcription or translation of one ormore nucleic acids.

In certain embodiments, antisense compounds specifically hybridize whenthere is a sufficient degree of complementarity to avoid non-specificbinding of the antisense compound to non-target nucleic acid sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In certain embodiments, oligomeric compounds of the present inventionare RNAi compounds. In certain embodiments, oligomeric compounds of thepresent invention are ssRNA compounds. In certain embodiments,oligomeric compounds of the present invention are paired with a secondoligomeric compound to form an siRNA. In certain such embodiments, thesecond oligomeric compound is also an oligomeric compound of the presentinvention. In certain embodiments, the second oligomeric compound is anymodified or unmodified nucleic acid. In certain embodiments, theoligomeric compound of the present invention is the antisense strand inan siRNA compound. In certain embodiments, the oligomeric compound ofthe present invention is the sense strand in an siRNA compound.

Single-Stranded Antisense Compounds

In certain embodiments, oligomeric compounds of the present inventionare particularly suited for use as single-stranded antisense compounds.In certain such embodiments, such oligomeric compounds aresingle-stranded RNAi compounds. In certain embodiments, such oligomericcompounds are ssRNA compounds or microRNA mimics. Certain 5′-terminalnucleosides described herein are suited for use in such single-strandedoligomeric compounds. In certain embodiments, such 5′-terminalnucleosides stabilize the 5′-phosphorous moiety. In certain embodiments,5′-terminal nucleosides of the present invention are resistant tonucleases. In certain embodiments, the motifs of the present inventionare particularly suited for use in single-stranded oligomeric compounds.

Use of single-stranded RNAi compounds has been limited. In certaininstances, single stranded RNAi compounds are quickly degraded and/or donot load efficiently into RISC. Certain compounds of the presentinvention possess properties superior to previously described ssRNAicompounds. In certain embodiments, oligomeric compounds of the presentinvention are superior ssRNAi compounds in vitro. In certain suchembodiments, the 5′-terminal phosphorous moiety is stabilized. Incertain such embodiments, the 5′-nucleoside is resistant to nucleasecleavage. In certain embodiments, the 5′-terminal end loads efficientlyinto RISC. In certain embodiments, the motif stabilizes the oligomericcompound. In certain embodiments the 3′-terminal end of the oligomericcompound is stabilized.

Design of single-stranded RNAi compounds for use in cells and/or for usein vivo presents several challenges. For example, the compound must bechemically stable, resistant to nuclease degradation, capable ofentering cells, capable of loading into RISC (e.g., binding Ago1 orAgo2), capable of hybridizing with a target nucleic acid, and not toxicto cells or animals. In certain instances, a modification or motif thatimproves one such feature may worsen another feature, rendering acompound having such modification or motif unsuitable for use as an RNAicompound. For example, certain modifications, particularly if placed ator near the 5′-end of an oligomeric compound, may make the compound morestable and more resistant to nuclease degradation, but may also inhibitor prevent loading into RISC by blocking the interaction with RISCcomponents, such as Ago1 or Ago2. Despite its improved stabilityproperties, such a compound would be unsuitable for use in RNAi. Thus,the challenge is to identify modifications and combinations andplacement of modifications that satisfy each parameter at leastsufficient to provide a functional single-stranded RNAi compound. Incertain embodiments, oligomeric compounds of the present inventioncombine modifications to provide single-stranded RNAi compounds that areactive as single-stranded RNAi compounds.

In certain instances, a single-stranded oligomeric compound comprising a5′-phosphorous moiety is desired. For example, in certain embodiments,such 5′-phosphorous moiety is necessary or useful for RNAi compounds,particularly, single-stranded RNAi compounds. In such instances, it isfurther desirable to stabilize the phosphorous moiety againstdegradation or de-phosphorylation which may inactivate the compound.Further, it is desirable to stabilize the entire 5′-nucleoside fromdegradation, which could also inactivate the compound. Thus, in certainembodiments, oligonucleotides in which both the 5′-phosphorous moietyand the 5′-nucleoside have been stabilized are desired. In certainembodiments, the present invention provides modified nucleosides thatmay be placed at the 5′-end of an oligomeric compound, resulting instabilized phosphorous and stabilized nucleoside. In certain suchembodiments, the phosphorous moiety is resistant to removal inbiological systems, relative to unmodified nucleosides and/or the5′-nucleoside is resistant to cleavage by nucleases. In certainembodiments, such nucleosides are modified at one, at two or at allthree of: the 2′-position, the 5′-position, and at the phosphorousmoiety. Such modified nucleosides may be incorporated at the 5′-end ofan oligomeric compound.

Although certain oligomeric compounds of the present invention haveparticular use as single-stranded compounds, such compounds may also bepaired with a second strand to create a double-stranded oligomericcompound. In such embodiments, the second strand of the double-strandedduplex may or may not also be an oligomeric compound of the presentinvention.

In certain embodiments, oligomeric compounds of the present inventionbind and/or activate one or more nucleases. In certain embodiments, suchbinding and/or activation ultimately results in antisense activity. Incertain embodiments, an oligomeric compound of the invention interactswith a target nucleic acid and with a nuclease, resulting in activationof the nuclease and cleavage of the target nucleic acid. In certainembodiments, an oligomeric compound of the invention interacts with atarget nucleic acid and with a nuclease, resulting in activation of thenuclease and inactivation of the target nucleic acid. In certainembodiments, an oligomeric compound of the invention forms a duplex witha target nucleic acid and that duplex activates a nuclease, resulting incleavage and/or inactivation of one or both of the oligomeric compoundand the target nucleic acid. In certain embodiments, an oligomericcompound of the invention binds and/or activates a nuclease and thebound and/or activated nuclease cleaves or inactivates a target nucleicacid. Nucleases include, but are not limited to, ribonucleases(nucleases that specifically cleave ribonucleotides), double-strandnucleases (nucleases that specifically cleave one or both strands of adouble-stranded duplex), and double-strand ribonucleases. For example,nucleases include, but are not limited to RNase H, an argonaute protein(including, but not limited to Ago2), and dicer.

In certain embodiments, oligomeric compounds of the present inventioninteract with an argonaute protein (Ago). In certain embodiments, sucholigomeric compounds first enter the RISC pathway by interacting withanother member of the pathway (e.g., dicer). In certain embodiments,oligomeric compounds first enter the RISC pathway by interacting withAgo. In certain embodiments, such interaction ultimately results inantisense activity. In certain embodiments, the invention providesmethods of activating Ago comprising contacting Ago with an oligomericcompound. In certain embodiments, such oligomeric compounds comprise amodified 5′-phosphate group. In certain embodiments, the inventionprovides methods of modulating the expression or amount of a targetnucleic acid in a cell comprising contacting the cell with an oligomericcompound capable of activating Ago, ultimately resulting in cleavage ofthe target nucleic acid. In certain embodiments, the cell is in ananimal. In certain embodiments, the cell is in vitro. In certainembodiments, the methods are performed in the presence of manganese. Incertain embodiments, the manganese is endogenous. In certain embodimentthe methods are performed in the absence of magnesium. In certainembodiments, the Ago is endogenous to the cell. In certain suchembodiments, the cell is in an animal. In certain embodiments, the Agois human Ago. In certain embodiments, the Ago is Ago2. In certainembodiments, the Ago is human Ago2.

In certain embodiments, oligomeric compounds of the present inventioninteract with the enzyme dicer. In certain such embodiments, oligomericcompounds bind to dicer and/or are cleaved by dicer. In certain suchembodiments, such interaction with dicer ultimately results in antisenseactivity. In certain embodiments, the dicer is human dicer. In certainembodiments, oligomeric compounds that interact with dicer aredouble-stranded oligomeric compounds. In certain embodiments, oligomericcompounds that interact with dicer are single-stranded oligomericcompounds.

In embodiments in which a double-stranded oligomeric compound interactswith dicer, such double-stranded oligomeric compound forms a dicerduplex. In certain embodiments, any oligomeric compound described hereinmay be suitable as one or both strands of a dicer duplex. In certainembodiments, each strand of the dicer duplex is an oligomeric compoundof the present invention. In certain embodiments, one strand of thedicer duplex is an oligomeric compound of the present invention and theother strand is any modified or unmodified oligomeric compound. Incertain embodiments, one or both strands of a dicer duplex comprises anucleoside of Formula II or IIa at the 5′ end. In certain embodiments,one strand of a dicer duplex is an antisense oligomeric compound and theother strand is its sense complement.

In certain embodiments, the dicer duplex comprises a 3′-overhang at oneor both ends. In certain embodiments, such overhangs are additionalnucleosides. In certain embodiments, the dicer duplex comprises a 3′overhang on the sense oligonucleotide and not on the antisenseoligonucleotide. In certain embodiments, the dicer duplex comprises a 3′overhang on the antisense oligonucleotide and not on the senseoligonucleotide. In certain embodiments, 3′ overhangs of a dicer duplexcomprise 1-4 nucleosides. In certain embodiments, such overhangscomprise two nucleosides. In certain embodiments, the nucleosides in the3′-overhangs comprise purine nucleobases. In certain embodiments, thenucleosides in the 3′ overhangs comprise adenine nucleobases. In certainembodiments, the nucleosides in the 3′ overhangs comprise pyrimidines.In certain embodiments, dicer duplexes comprising 3′-purine overhangsare more active as antisense compounds than dicer duplexes comprising 3′pyrimidine overhangs. In certain embodiments, oligomeric compounds of adicer duplex comprise one or more 3′ deoxy nucleosides. In certain suchembodiments, the 3′ deoxy nucleosides are dT nucleosides.

In certain embodiments, the 5′ end of each strand of a dicer duplexcomprises a phosphate moiety. In certain embodiments the antisensestrand of a dicer duplex comprises a phosphate moiety and the sensestrand of the dicer duplex does not comprise a phosphate moiety. Incertain embodiments the sense strand of a dicer duplex comprises aphosphate moiety and the antisense strand of the dicer duplex does notcomprise a phosphate moiety. In certain embodiments, a dicer duplex doesnot comprise a phosphate moiety at the 3′ end. In certain embodiments, adicer duplex is cleaved by dicer. In such embodiments, dicer duplexes donot comprise 2′-OMe modifications on the nucleosides at the cleavagesite. In certain embodiments, such cleavage site nucleosides are RNA.

In certain embodiments, interaction of an oligomeric compound with dicerultimately results in antisense activity. In certain embodiments, dicercleaves one or both strands of a double-stranded oligomeric compound andthe resulting product enters the RISC pathway, ultimately resulting inantisense activity. In certain embodiments, dicer does not cleave eitherstrand of a double-stranded oligomeric compound, but neverthelessfacilitates entry into the RISC pathway and ultimately results inantisense activity. In certain embodiments, dicer cleaves asingle-stranded oligomeric compound and the resulting product enters theRISC pathway, ultimately resulting in antisense activity. In certainembodiments, dicer does not cleave the single-stranded oligomericcompound, but nevertheless facilitates entry into the RISC pathway andultimately results in antisense activity.

In certain embodiments, the invention provides methods of activatingdicer comprising contacting dicer with an oligomeric compound. Incertain such embodiments, the dicer is in a cell. In certain suchembodiments, the cell is in an animal.

Dicer

In certain embodiments, oligomeric compounds of the present inventioninteract with the enzyme dicer. In certain such embodiments, oligomericcompounds bind to dicer and/or are cleaved by dicer. In certain suchembodiments, such interaction with dicer ultimately results in antisenseactivity. In certain embodiments, the dicer is human dicer. In certainembodiments, oligomeric compounds that interact with dicer aredouble-stranded oligomeric compounds. In certain embodiments, oligomericcompounds that interact with dicer are single-stranded oligomericcompounds.

In embodiments in which a double-stranded oligomeric compound interactswith dicer, such double-stranded oligomeric compound forms a dicerduplex. In certain embodiments, any oligomeric compound described hereinmay be suitable as one or both strands of a dicer duplex. In certainembodiments, each strand of the dicer duplex is an oligomeric compoundof the present invention. In certain embodiments, one strand of thedicer duplex is an oligomeric compound of the present invention and theother strand is any modified or unmodified oligomeric compound. Incertain embodiments, one or both strands of a dicer duplex comprises anucleoside of Formula II or IIa at the 5′. In certain embodiments, onestrand of a dicer duplex is an antisense oligomeric compound and theother strand is its sense complement.

In certain embodiments, the invention provides single-strandedoligomeric compounds that interact with dicer. In certain embodiments,such single-stranded dicer compounds comprise a nucleoside of Formula IIor IIa. In certain embodiments, single-stranded dicer compounds do notcomprise a phosphorous moiety at the 3′-end. In certain embodiments,such single-stranded dicer compounds may comprise a 3′-overhangs. Incertain embodiments, such 3′-overhangs are additional nucleosides. Incertain embodiments, such 3′-overhangs comprise 1-4 additionalnucleosides that are not complementary to a target nucleic acid and/orare differently modified from the adjacent 3′ nucleoside of theoligomeric compound. In certain embodiments, a single-strandedoligomeric compound comprises an antisense oligonucleotide having two3′-end overhang nucleosides wherein the overhang nucleosides are adenineor modified adenine nucleosides. In certain embodiments, single strandedoligomeric compounds that interact with dicer comprise a nucleoside ofFormula II or IIa

In certain embodiments, interaction of an oligomeric compound with dicerultimately results in antisense activity. In certain embodiments, dicercleaves one or both strands of a double-stranded oligomeric compound andthe resulting product enters the RISC pathway, ultimately resulting inantisense activity. In certain embodiments, dicer does not cleave eitherstrand of a double-stranded oligomeric compound, but neverthelessfacilitates entry into the RISC pathway and ultimately results inantisense activity. In certain embodiments, dicer cleaves asingle-stranded oligomeric compound and the resulting product enters theRISC pathway, ultimately resulting in antisense activity. In certainembodiments, dicer does not cleave the single-stranded oligomericcompound, but nevertheless facilitates entry into the RISC pathway andultimately results in antisense activity.

In certain embodiments, the invention provides methods of activatingdicer comprising contacting dicer with an oligomeric compound. Incertain such embodiments, the dicer is in a cell. In certain suchembodiments, the cell is in an animal.

Ago

In certain embodiments, oligomeric compounds of the present inventioninteract with Ago. In certain embodiments, such oligomeric compoundsfirst enter the RISC pathway by interacting with another member of thepathway (e.g., dicer). In certain embodiments, oligomeric compoundsfirst enter the RISC pathway by interacting with Ago. In certainembodiments, such interaction ultimately results in antisense activity.In certain embodiments, the invention provides methods of activating Agocomprising contacting Ago with an oligomeric compound. In certain suchembodiments, the Ago is in a cell. In certain such embodiments, the cellis in an animal.

Oligomeric Compound Identity

In certain embodiments, a portion of an oligomeric compound is 100%identical to the nucleobase sequence of a microRNA, but the entireoligomeric compound is not fully identical to the microRNA. In certainsuch embodiments, the length of an oligomeric compound having a 100%identical portion is greater than the length of the microRNA. Forexample, a microRNA mimic consisting of 24 linked nucleosides, where thenucleobases at positions 1 through 23 are each identical tocorresponding positions of a microRNA that is 23 nucleobases in length,has a 23 nucleoside portion that is 100% identical to the nucleobasesequence of the microRNA and has approximately 96% overall identity tothe nucleobase sequence of the microRNA.

In certain embodiments, the nucleobase sequence of oligomeric compoundis fully identical to the nucleobase sequence of a portion of amicroRNA. For example, a single-stranded microRNA mimic consisting of 22linked nucleosides, where the nucleobases of positions 1 through 22 areeach identical to a corresponding position of a microRNA that is 23nucleobases in length, is fully identical to a 22 nucleobase portion ofthe nucleobase sequence of the microRNA. Such a single-stranded microRNAmimic has approximately 96% overall identity to the nucleobase sequenceof the entire microRNA, and has 100% identity to a 22 nucleobase portionof the microRNA.

Synthesis of Monomeric and Oligomeric Compounds

The nucleosides provided herein can be prepared by any of the applicabletechniques of organic synthesis, as, for example, illustrated in theexamples below. Many such techniques are well known in the art. However,many of the known techniques are elaborated in Compendium of OrganicSynthetic Methods, John Wiley & Sons, New York: Vol. 1, Ian T. Harrisonand Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison,1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G.Wade Jr., 1980; Vol. 5, Leroy G. Wade Jr., 1984; and Vol. 6, Michael B.Smith; as well as March, J., Advanced Organic Chemistry, 3rd Edition,John Wiley & Sons, New York, 1985; Comprehensive Organic Synthesis.Selectivity, Strategy & Efficiency in Modern Organic Chemistry, in 9Volumes, Barry M. Trost, Editor-in-Chief, Pergamon Press, New York,1993; Advanced Organic Chemistry, Part B: Reactions and Synthesis, 4thEdition; Carey and Sundberg, Kluwer Academic/Plenum Publishers, NewYork, 2001; Advanced Organic Chemistry, Reactions, Mechanisms, andStructure, 2nd Edition, March, McGraw Hill, 1977; Greene, T. W., andWutz, P. G. M., Protecting Groups in Organic Synthesis, 4th Edition,John Wiley & Sons, New York, 1991; and Larock, R. C., ComprehensiveOrganic Transformations, 2nd Edition, John Wiley & Sons, New York, 1999.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, α or β, or as (D)- or (L)- such as foramino acids. Included herein are all such possible isomers, as well astheir racemic and optically pure forms. Optical isomers may be preparedfrom their respective optically active precursors by the proceduresdescribed above, or by resolving the racemic mixtures. The resolutioncan be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions, John Wiley & Sons, 1981. Whenthe compounds described herein contain olefinic double bonds, otherunsaturation, or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers or cis- and trans-isomers. Likewise, alltautomeric forms are also intended to be included. The configuration ofany carbon-carbon double bond appearing herein is selected forconvenience only and is not intended to limit a particular configurationunless the text so states.

In certain embodiments, the preparation of oligomeric compounds asdisclosed herein is performed according to literature procedures forDNA: Protocols for Oligonucleotides and Analogs, Agrawal, Ed., HumanaPress, 1993, and/or RNA: Scaringe, Methods, 2001, 23, 206-217; Gait etal., Applications of Chemically synthesized RNA in RNA:ProteinInteractions, Smith, Ed., 1998, 1-36; Gallo et al., Tetrahedron, 2001,57, 5707-5713. Additional methods for solid-phase synthesis may be foundin Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777;4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re.34,069.

Synthesis of Oligomeric Compounds

Oligomeric compounds are routinely prepared using solid support methodsas opposed to solution phase methods. Commercially available equipmentcommonly used for the preparation of oligomeric compounds that utilizethe solid support method is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed. Suitable solid phase techniques, including automated synthesistechniques, are described in Oligonucleotides and Analogues, a PracticalApproach, F. Eckstein, Ed., Oxford University Press, New York, 1991.

The synthesis of RNA and related analogs relative to the synthesis ofDNA and related analogs has been increasing as efforts in RNAinterference and micro RNA increase. The primary RNA synthesisstrategies that are presently being used commercially include5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS),5′-O-DMT-2′-O—[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP),2′-O—[(triisopropylsilyl)oxy]methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM) and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries. The primary groups being used for commercial RNA synthesisare: TBDMS: 5′-O-DMT-2′-O-t-butyldimethylsilyl; TOM:2′-O—[(triisopropylsilyl)oxy]methyl; DOD/ACE:(5′-O-bis(trimethylsiloxy)cyclo-dodecyloxysilylether-2′-O-bis(2-acetoxyethoxy)methyl; and FPMP:5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-ethoxypiperidin-4-yl]. In certainembodiments, each of the aforementioned RNA synthesis strategies can beused herein. In certain embodiments, the aforementioned RNA synthesisstrategies can be performed together in a hybrid fashion e.g. using a5′-protecting group from one strategy with a 2′-O-protecting fromanother strategy.

Compositions and Methods for Formulating Pharmaceutical Compositions

Oligomeric compounds may be admixed with pharmaceutically acceptableactive and/or inert substances for the preparation of pharmaceuticalcompositions or formulations. Compositions and methods for theformulation of pharmaceutical compositions are dependent upon a numberof criteria, including, but not limited to, route of administration,extent of disease, or dose to be administered.

Oligomeric compounds, including antisense compounds, can be utilized inpharmaceutical compositions by combining such oligomeric compounds witha suitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in certain embodiments, employed in themethods described herein is a pharmaceutical composition comprising anantisense compound and a pharmaceutically acceptable diluent. In certainembodiments, the pharmaceutically acceptable diluent is PBS.

Pharmaceutical compositions comprising oligomeric compounds encompassany pharmaceutically acceptable salts, esters, or salts of such esters.In certain embodiments, pharmaceutical compositions comprisingoligomeric compounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active oligomeric compound.

Lipid-based vectors have been used in nucleic acid therapies in avariety of methods. In one method, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In another method, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.

In certain methods, preparations are made that include a polyaminecompound or a lipid moiety complexed with a nucleic acid. Suchpreparations are described in PCT publication WO/2008/042973; and inAkinc et al., Nature Biotechnology 26, 561-569 (1 May 2008), which areherein incorporated by reference in their entirety.

Certain Methods/Uses

In certain embodiments, the present invention provides compounds andmethods for reducing the amount or activity of a target nucleic acid. Incertain embodiments, the invention provides antisense compounds andmethods. In certain embodiments, the invention provides antisensecompounds and methods based on activation of RNase H. In certainembodiments, the invention provides RNAi compounds and methods.

In certain instances it is desirable to use an antisense compound thatfunctions at least in part through RISC. In certain such instancesunmodified RNA, whether single-stranded or double stranded is notsuitable. Single-stranded RNA is relatively unstable and double-strandedRNA does not easily enter cells. The challenge has been to identifymodifications and motifs that provide desirable properties, such asimproved stability, without interfering with (and possibly evenimproving upon) the antisense activity of RNA through RNAi.

In certain embodiments, the present invention provides oligonucleotideshaving motifs (nucleoside motifs and/or linkage motifs) that result inimproved properties. Certain such motifs result in single-strandedoligonucleotides with improved stability and/or cellular uptakeproperties while retaining antisense activity. For example,oligonucleotides having an alternating nucleoside motif and sevenphosphorothioate linkages at to 3′-terminal end have improved stabilityand activity. Similar compounds that comprise phosphorothioate linkagesat each linkage have further improved stability, but are not active asRNAi compounds, presumably because the additional phosphorothioatelinkages interfere with the interaction of the oligonucleotide with theRISC pathway components (e.g., with Ago). In certain embodiments, theoligonucleotides having motifs herein result in single-stranded RNAicompounds having desirable properties. In certain embodiments, sucholigonucleotides may be paired with a second strand to form adouble-stranded RNAi compound. In such embodiments, the second strand ofsuch double-stranded RNAi compounds may comprise a motif of the presentinvention, may comprise another motif of modifications or may beunmodified.

It has been shown that in certain circumstances for single-stranded RNAcomprising a 5′-phosphate group has RNAi activity if but has much lessRNAi activity if it lacks such 5′-phosphate group. The present inventorshave recognized that in certain circumstances unmodified 5′-phophategroups may be unstable (either chemically or enzymatically).Accordingly, in certain circumstances, it is desirable to modify theoligonucleotide to stabilize the 5′-phosphate. In certain embodiments,this is achieved by modifying the phosphate group. In certainembodiments, this is achieved by modifying the sugar of the 5′-terminalnucleoside. In certain embodiments, this is achieved by modifying thephosphate group and the sugar. In certain embodiments, the sugar ismodified at the 5′-position, the 2′-position, or both the 5′-positionand the 2′-position. As with motifs, above, in embodiments in which RNAiactivity is desired, a phosphate stabilizing modification must notinterfere with the ability of the oligonucleotide to interact with RISCpathway components (e.g., with Ago).

In certain embodiments, the invention provides oligonucleotidescomprising a phosphate-stabilizing modification and a motif describedherein. In certain embodiments, such oligonucleotides are useful assingle-stranded RNAi compounds having desirable properties. In certainembodiments, such oligonucleotides may be paired with a second strand toform a double-stranded RNAi compound. In such embodiments, the secondstrand may comprise a motif of the present invention, may compriseanother motif of modifications or may be unmodified RNA.

The target for such antisense compounds comprising a motif and/or5′-phosphate stabilizing modification of the present invention can beany naturally occurring nucleic acid. In certain embodiments, the targetis selected from: pre-mRNA, mRNA, non-coding RNA, small non-coding RNA,pd-RNA, and microRNA. In embodiments, in which a target nucleic acid isa pre-RNA or a mRNA, the target may be the same as that of a naturallyoccurring micro-RNA (i.e., the oligonucleotide may be a microRNA mimic).In such embodiments, there may be more than one target mRNA.

In certain embodiments, the invention provides compounds and methods forantisense activity in a cell. In certain embodiments, the cell is in ananimal. In certain embodiments, the animal is a human. In certainembodiments, the invention provides methods of administering a compoundof the present invention to an animal to modulate the amount or activityor function of one or more target nucleic acid.

In certain embodiments oligonucleotides comprise one or more motifs ofthe present invention, but do not comprise a phosphate stabilizingmodification. In certain embodiments, such oligonucleotides are usefulfor in vitro applications. In certain embodiments, such oligonucleotidesare useful for in vivo applications where RISC activity is not required.For example, in certain embodiments, such oligonucleotides altersplicing of pre-mRNA.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

EXAMPLES General

¹H and ¹³C NMR spectra were recorded on a 300 MHz and 75 MHz Brukerspectrometer, respectively.

Example 1 Synthesis of Nucleoside Phosphoramidites

The preparation of nucleoside phosphoramidites is performed followingprocedures that are illustrated herein and in the art such as but notlimited to U.S. Pat. No. 6,426,220 and published PCT WO 02/36743.

Example 2 Synthesis of Oligomeric Compounds

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as alkylatedderivatives and those having phosphorothioate linkages.

Oligomeric compounds: Unsubstituted and substituted phosphodiester (P=O)oligomeric compounds, including without limitation, oligonucleotides canbe synthesized on an automated DNA synthesizer (Applied Biosystems model394) using standard phosphoramidite chemistry with oxidation by iodine.

In certain embodiments, phosphorothioate internucleoside linkages (P=S)are synthesized similar to phosphodiester internucleoside linkages withthe following exceptions: thiation is effected by utilizing a 10% w/vsolution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile forthe oxidation of the phosphite linkages. The thiation reaction step timeis increased to 180 sec and preceded by the normal capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (12-16 hr), the oligomeric compounds are recoveredby precipitating with greater than 3 volumes of ethanol from a 1 MNH₄OAc solution. Phosphinate internucleoside linkages can be prepared asdescribed in U.S. Pat. No. 5,508,270.

Alkyl phosphonate internucleoside linkages can be prepared as describedin U.S. Pat. No. 4,469,863.

3′-Deoxy-3′-methylene phosphonate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.

Phosphoramidite internucleoside linkages can be prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.

Alkylphosphonothioate internucleoside linkages can be prepared asdescribed in published PCT applications PCT/US94/00902 andPCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,476,925.

Phosphotriester internucleoside linkages can be prepared as described inU.S. Pat. No. 5,023,243.

Borano phosphate internucleoside linkages can be prepared as describedin U.S. Pat. Nos. 5,130,302 and 5,177,198.

Oligomeric compounds having one or more non-phosphorus containinginternucleoside linkages including without limitationmethylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides,methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P=O or P=S linkages can be prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal internucleoside linkages can be preparedas described in U.S. Pat. Nos. 5,264,562 and 5,264,564.

Ethylene oxide internucleoside linkages can be prepared as described inU.S. Pat. No. 5,223,618.

Example 3 Isolation and Purification of Oligomeric Compounds

After cleavage from the controlled pore glass solid support or othersupport medium and deblocking in concentrated ammonium hydroxide at 55°C. for 12-16 hours, the oligomeric compounds, including withoutlimitation oligonucleotides and oligonucleosides, are recovered byprecipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligomeric compounds are analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresis.The relative amounts of phosphorothioate and phosphodiester linkagesobtained in the synthesis is determined by the ratio of correctmolecular weight relative to the −16 amu product (+/−32+/−48). For somestudies oligomeric compounds are purified by HPLC, as described byChiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtainedwith HPLC-purified material are generally similar to those obtained withnon-HPLC purified material.

Example 4 Synthesis of Oligomeric Compounds Using the 96 Well PlateFormat

Oligomeric compounds, including without limitation oligonucleotides, canbe synthesized via solid phase P(III) phosphoramidite chemistry on anautomated synthesizer capable of assembling 96 sequences simultaneouslyin a 96-well format. Phosphodiester internucleoside linkages areafforded by oxidation with aqueous iodine. Phosphorothioateinternucleoside linkages are generated by sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrousacetonitrile. Standard base-protected beta-cyanoethyl-diiso-propylphosphoramidites can be purchased from commercial vendors (e.g.PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway,N.J.). Non-standard nucleosides are synthesized as per standard orpatented methods and can be functionalized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligomeric compounds can be cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 5 Analysis of Oligomeric Compounds Using the 96-Well PlateFormat

The concentration of oligomeric compounds in each well can be assessedby dilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products can be evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates are diluted fromthe master plate using single and multi-channel robotic pipettors.Plates are judged to be acceptable if at least 85% of the oligomericcompounds on the plate are at least 85% full length.

Example 6 In Vitro Treatment of Cells with Oligomeric Compounds

The effect of oligomeric compounds on target nucleic acid expression istested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Cell linesderived from multiple tissues and species can be obtained from AmericanType Culture Collection (ATCC, Manassas, Va.).

The following cell type is provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays or RT-PCR.

b.END cells: The mouse brain endothelial cell line b.END was obtainedfrom Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany).b.END cells are routinely cultured in DMEM, high glucose (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells areroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells are seeded into 96-well plates(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a densityof approximately 3000 cells/well for uses including but not limited tooligomeric compound transfection experiments.

Experiments involving treatment of cells with oligomeric compounds:

When cells reach appropriate confluency, they are treated witholigomeric compounds using a transfection method as described.

Lipofectin™

When cells reached 65-75% confluency, they are treated with one or moreoligomeric compounds. The oligomeric compound is mixed with LIPOFECTIN™Invitrogen Life Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reducedserum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achievethe desired concentration of the oligomeric compound(s) and aLIPOFECTIN™ concentration of 2.5 or 3 μg/mL per 100 nM oligomericcompound(s). This transfection mixture is incubated at room temperaturefor approximately 0.5 hours. For cells grown in 96-well plates, wellsare washed once with 100 μl, OPTI-MEM™-1 and then treated with 130 μL ofthe transfection mixture. Cells grown in 24-well plates or otherstandard tissue culture plates are treated similarly, using appropriatevolumes of medium and oligomeric compound(s). Cells are treated and dataare obtained in duplicate or triplicate. After approximately 4-7 hoursof treatment at 37° C., the medium containing the transfection mixtureis replaced with fresh culture medium. Cells are harvested 16-24 hoursafter treatment with oligomeric compound(s).

Other suitable transfection reagents known in the art include, but arenot limited to, CYTOFECTIN™, LIPOFECTAMINE™, OLIGOFECTAMINE™, andFUGENE™. Other suitable transfection methods known in the art include,but are not limited to, electroporation.

Example 7 Real-Time Quantitative PCR Analysis of Target mRNA Levels

Quantitation of target mRNA levels is accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

RT and PCR reagents are obtained from Invitrogen Life Technologies(Carlsbad, Calif.). RT, real-time PCR is carried out by adding 20 μL PCRcocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP,dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer,125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction iscarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol are carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR are normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RIBOGREEN™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RIBOGREEN™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RIBOGREEN™ working reagent (RIBOGREEN™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Example 8 Analysis of Oligonucleotide Inhibition of Target Expression

Antisense modulation of a target expression can be assayed in a varietyof ways known in the art. For example, a target mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently desired. RNA analysis can be performed on total cellular RNAor poly(A)+ mRNA. One method of RNA analysis of the present disclosureis the use of total cellular RNA as described in other examples herein.Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISMT™7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Protein levels of a target can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art. Methods for preparation ofpolyclonal antisera are taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9,John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 9 Design of Phenotypic Assays and In Vivo Studies for the Use ofTarget Inhibitors Phenotypic Assays

Once target inhibitors have been identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with atarget inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Measurement of the expression of one or more of the genes of the cellafter treatment is also used as an indicator of the efficacy or potencyof the a target inhibitors. Hallmark genes, or those genes suspected tobe associated with a specific disease state, condition, or phenotype,are measured in both treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

Example 10 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA is isolated according to Miura et al., (Clip. Chem., 1996,42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine inthe art. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, theplate is gently agitated and then incubated at room temperature for fiveminutes. 55 μl, of lysate is transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates are incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plateis blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C., is added to each well, the plate is incubated on a90° C. hot plate for 5 minutes, and the eluate is then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA is isolated using an RNEASY 96™ kit and buffers purchased fromQiagen Inc. (Valencia, Calif.) following the manufacturer's recommendedprocedures. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 150μL Buffer RLT is added to each well and the plate vigorously agitatedfor 20 seconds. 150 μL of 70% ethanol is then added to each well and thecontents mixed by pipetting three times up and down. The samples arethen transferred to the RNEASY 96™ well plate attached to a QIAVAC™manifold fitted with a waste collection tray and attached to a vacuumsource. Vacuum is applied for 1 minute. 500 μL of Buffer RW1 is added toeach well of the RNEASY 96™ plate and incubated for 15 minutes and thevacuum is again applied for 1 minute. An additional 500 μL of Buffer RW1is added to each well of the RNEASY 96™ plate and the vacuum is appliedfor 2 minutes. 1 mL of Buffer RPE is then added to each well of theRNEASY 96™ plate and the vacuum applied for a period of 90 seconds. TheBuffer RPE wash is then repeated and the vacuum is applied for anadditional 3 minutes. The plate is then removed from the QIAVAC™manifold and blotted dry on paper towels. The plate is then re-attachedto the QIAVAC™ manifold fitted with a collection tube rack containing1.2 mL collection tubes. RNA is then eluted by pipetting 140 μL of RNAsefree water into each well, incubating 1 minute, and then applying thevacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 11 Target-Specific Primers and Probes

Probes and primers may be designed to hybridize to a target sequence,using published sequence information.

For example, for human PTEN, the following primer-probe set was designedusing published sequence information (GENBANK™ accession numberU92436.1, SEQ ID NO: 1).

Forward primer: (SEQ ID NO: 2) AATGGCTAAGTGAAGATGACAATCATReverse primer: (SEQ ID NO: 3) TGCACATATCATTACACCAGTTCGTAnd the PCR probe:

FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4), where FAM isthe fluorescent dye and TAMRA is the quencher dye.

Example 12 Western Blot Analysis of Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to a target is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 13 Preparation of Compound 5

Compound 1 is prepared according to the procedures published in U.S.Pat. No. 5,969,116.

Example 14 Preparation of Compound 13

a) Preparation of Compound 7

Compound 6 was prepared according to the method of De Mesmaeker whereinNapBr was used instead of BnBr (Mesmaeker et al., Synlett, 1997,1287-1290). Dried Compound 6 (21.1 g, 47.04 mmol) was dissolved in amixture of glacial acetic acid (104 mL) and acetic anhydride (17.2 mL).To this solution was added 14 drops of concentrated H₂SO₄. After 1.5 h,the resulting light brown solution was diluted in EtOAc (600 mL), washedwith sat. NaHCO₃ (5×600 mL), dried over anhydrous Na₂SO₄, filtered,evaporated and dried under high vacuum to yield Compound 7 (22.7 g, 99%)as a pale oil. ES MS m/z 515.1 [M+Na]⁺.

b) Preparation of Compound 8

A mixture of Compound 7 (23.3 g, 46.70 mmol) and thymine (10.01 g, 79.40mmol) was suspended in anhydrous CH₃CN (233 mL). To this mixture wasadded N,O-bis-trimethylsilylacetamide (41.06 mL, 167.94 mmol), followedby heating at 55° C. for 1 h. The mixture was cooled to 0° C., thentrimethylsilyl trifluoromethanesulfonate (19.07 mL, 105.54 mmol) wasadded dropwise over 15 min. The mixture was subsequently heated at 55°C. After 3 hours the mixture was cooled to 0° C. and quenched with thedropwise addition of saturated aqueous NaHCO₃ (20 mL). The mixture waspoured into EtOAc, washed with brine (4×0.8 mL), dried over anhydrousNa₂SO₄, filtered, evaporated and dried under high vacuum. The residuewas purified by silica gel column chromatography and eluted with 20% to50% EtOAc in hexanes to yield Compound 8 (22.27 g, 85%) as a white foam.ES MS m/z 559.2 [M+H]⁺.

c) Preparation of Compound 9

Compound 8 (11.71 g, 20.98 mmol) was dissolved in anhydrous DMF (115mL). To this was added 1,8-diazabicycl-[5-4-0]undec-7-ene (DBU, 9.30 mL,62.41 mmol). The reaction mixture was cooled in an ice bath. To this wasadded benzyl chloromethyl ether (4.36 mL, 31.47 mmol), and stirred at 0°C. for 1 hour. The mixture was diluted with EtOAc (200 mL), washed withsaturated aqueous NaHCO₃ (200 mL) and brine (200 mL) then dried(Na₂SO₄), filtered and evaporated. The residue obtained was dissolved inmethanol (89 mL) and K₂CO₃ (8.76 g, 63.40 mmol). The reaction mixturewas stirred at room temperature for 1 h. The mixture was poured intoEtOAc (200 mL), washed with water (200 mL) and brine (200 mL), driedover anhydrous Na₂SO₄, filtered and evaporated. The residue was purifiedby silica gel column chromatography and eluted with 5% methanol inCH₂Cl₂ to yield Compound 9 (8.93 g, 80%) as a white foam. ES MS m/z533.2 [M+H]⁺.

d) Preparation of Compound 10

Compound 9 (4.30 g, 8.07 mmol) was dried over P₂O₅ under reducedpressure and dissolved in anhydrous DMF (24 mL). The mixture was cooledto −20° C. To this was added NaH (0.48 g, 12.11 mmol, 60% dispersion inmineral oil) with stirring for 30 minutes followed by addition of1-methoxy-2-iodoethane (2.25 g, 12.11 mmol). The reaction mixture waswarmed up to 0° C. After stirring for 1.5 h at 0° C. the reactionmixture was cooled to −20° C. and additional NaH (0.48 g, 12.11 mmol,60% dispersion in mineral oil) was added. Stirring was continued at −20°C. for 30 minutes and 1-methoxy-2-iodoethane (2.25 g, 12.11 mmol) wasadded. The reaction mixture was warmed to 0° C. and with stirring for anadditional 1.5 h. The reaction was quenched with methanol (5 mL),diluted with EtOAc (100 mL), washed with water (100 mL) and brine (100mL), dried over Na₂SO₄, filtered and evaporated under reduced pressure.The residue was purified by silica gel column chromatography and elutedwith 5% methanol in CH₂Cl₂ to yield Compound 10 (2.95 g, 62%). ES MS m/z591.2 [M+H]³⁰ .

e) Preparation of Compound 11

Compound 10 (2.2 g, 3.73 mmol) was dissolved in anhydrous pyridine (7mL) and cooled in an ice bath. To this benzoyl chloride (0.88 mL, 7.61mmol) was added and once the addition was over, reaction mixture wasallowed to come to room temperature. The reaction mixture was stirred atroom temperature for 4 h under an argon atmosphere and subsequentlycooled the reaction mixture in an ice bath and quenched by addingsaturated aqueous NaHCO₃ (5 mL). Diluted the reaction mixture with EtOAc(50 mL) and washed with saturated aqueous NaHCO₃ (2×50 mL), brine (50mL), dried over Na₂SO₄, filtered and concentrated. The residue obtainedwas dissolved in CH₂Cl₂ (40 mL) and added2,4-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 1.93 g, 8.5 mmol) andH₂O (0.15 mL, 8.5 mmol) and stirred at room temperature. After 18 h,diluted the reaction mixture with EtOAc (60 mL), washed with saturatedaqueous NaHCO₃ (2×80 mL), brine (50 mL), dried over Na₂SO₄, filtered andevaporated under reduced pressure. The residue was dissolved in MeOH (30mL) and palladium hydroxide (1.1 g, 20 wt % Pd on carbon dry base) andstirred under H₂ atmosphere for 6 h. To this acetic acid (0.56 mL) wasadded and stirred for 5 min. The reaction mixture was filtered through apad of celite 545, and washed the celite with copious amount of MeOH.The combined filtrate and washing were concentrated under reducedpressure and the residue was purified by silica gel columnchromatography and eluted with 5% methanol in CH₂Cl₂ to yield Compound11 (1.43 g, 88%). ES MS m/z 435.1 [M+H]⁺.

f) Preparation of Compound 12

A mixture of Compound II (1.33 g, 3.06 mmol) and imidazole (2.09, 30.70mmol) was dissolved in anhydrous DMF (11.4 mL). To this solutiontert-butyldimethylsilyl chloride (2.31 g, 15.33 mmol) was added withstirring at room temperature for 16 h under an atmosphere of argon. Thereaction mixture was diluted with EtOAc (75 mL) and washed withsaturated aqueous NaHCO₃ (2×60 mL) and brine (50 mL), dried over Na₂SO₄,filtered and concentrated. The residue obtained was dissolved inmethanolic ammonia (20 mL, 7M) and stirred for 24 h at 55° C. Thesolvent was removed under reduced pressure and the residue was purifiedby silica gel column chromatography and eluted with 50% EtOAc in hexanesto yield Compound 12 (1.21 g, 89%). ES MS m/z 455.2 [M+H]⁺.

g) Preparation of Compound 13

Compound 12 (0.42 g, 0.96 mmol) was mixed with 4,4′-dimethoxytritylchloride (0.82 g, 2.41 mmol) and dried over P₂O₅ under reduced pressure.The mixture was dissolved in anhydrous pyridine (3 mL) and stirred at45° C. for 18 h under an atmosphere of argon. The reaction mixture wascooled to room temperature and diluted with EtOAc (40 mL) and washedwith saturated aqueous NaHCO₃ (60 mL) and brine (40 mL), dried overNa₂SO₄, filtered and concentrated. The residue obtained was purified bysilica gel column chromatography and eluted first with 50% EtOAc inhexanes and then with 5% methanol in CH₂Cl₂. The product obtained wasdissolved in a mixture of triethylamine trihydrofluoride (1.38 mL, 8.44mmol) and triethylamine (0.58 mL, 4.22 mmol) in THF (8.4 mL). After 72 hthe mixture was diluted with EtOAc (60 mL), washed with water (40 mL),saturated aqueous NaHCO₃ (40 mL) and brine (40 mL) then dried overNa₂SO₄, filtered and evaporated. The residue obtained was purified bysilica gel column chromatography and eluted with 70% EtOAc in hexanes toyield Compound 13 (0.44 g, 73%). ES MS m/z 631.2 [M+H]⁺.

Example 15 Preparation of Compound 17

Compound 13 was prepared as per the procedures illustrated in Example14.

Example 16 Preparation of Compound 19

Compound 12 is prepared as per the procedures illustrated in Example 14.

Example 17 Preparation of Compounds 29-31

Compounds 1, 1a, 12 and 19 are prepared as per the proceduresillustrated in Examples 13, 17, 14 and 16, respectively. Compounds 20-22are prepared according to published procedures by Cook, J. Am. Chem.Soc., 1970, 92, 190-195.

Example 18 Preparation of Compounds 35-37

Compounds 1a, 12 and 19 are prepared as per the procedures illustratedin Examples 17, 14 and 16, respectively. Compounds 32-34 are preparedaccording to published procedures by Vincent et al., J. Org. Chem.,1998, 63, 7244-7257.

Example 19 Preparation of Compound 40

Compound 15 is prepared as per the procedures illustrated in Examples15.

Example 20 Oligomeric Compounds

Following synthetic procedures well known in the art, some of which areillustrated herein, oligomeric compounds are prepared having at leastone modified 5′ diphosphate nucleoside of Formula II or IIa, using oneor more of the phosphoramidite compounds illustrated in the precedingexamples (Example 13, Compound 5, Example 15, Compound 17, Example 17,Compounds 29, 30 and 31, Example 18, Compounds 35, 36 and 37, andExample 19, Compound 40).

Example 21 General Method for the Preparation of 5′ DiphosphateOligonucleotides Using Solid Phase Synthesis

Unless otherwise stated, all reagents and solutions used foroligonucleotide synthesis were purchased from commercial sources.

a) Preparation of Solid Supported Oligonucleotides 42

The 3′-di-isopropylphosphoramidites 41 bearing fast labile nucleobaseprotecting groups (i.e. Ac for C, Pac for A and iPrPac for G) werecommercially available from Chem Genes or custom synthesized by RXIChemicals. The oligonucleotides 42 were synthesized on a CPG solidsupport (Glen Research) using standard automated oligonucleotidesynthesis on an ABI394 (Applied Biosystems) synthesizer on the 40 μmolscale. The standard synthesis cycle was used for the following steps,including detritylation, phosphoramidite coupling, oxidation(sulfurization) and capping. The 3′-di-isopropylphosphoramidites 41 wereused as a 0.2 M solution in anhydrous acetonitrile (Glen Research).Capping A reagent was a Pac₂O solution. A Trityl-Off synthesis wasperformed to remove the last DMT group at the end of the synthesis. Theoligonucleotides were then washed with anhydrous acetonitrile followedby reverse flushed with argon. The resulting solid supportedoligonucleotides 42 were then stocked at −20° C.

B) Preparation of 5′-H-Phosphonate Monoester Oligonucleotides 44

0.25 to 2 μmol of solid supported oligonucleotides 42 (10 to 50 mg) wereplaced in a dry Twist oligonucleotide synthesis column (Glen Research).The column was closed and flushed with argon. A 1 M pyridine solution ofdiphenyl phosphite, which contained a mixture of 0.4 mL of diphenylphosphite and 1.6 mL of anhydrous pyridine (Aldrich) was gently pushedthrough the synthetic column back and forth for 30 minutes at roomtemperature. The column was then emptied and washed thoroughly withacetonitrile followed by reverse flushed with argon. A 100 mM aqueousTEAB (Aldrich) was pushed through the column for 2 hours after which thecolumn was then emptied, washed with anhydrous acetonitrile and reverseflushed with argon. The resulting H-phosphonate monoesteroligonucleotides 44 were placed under vacuum over P₂O₅ for 24 hours andstocked at −20° C.

C) Preparation of Solid Supported Phosphorimidazolidates 46

Solid supported H-phosphonate monoester oligonucleotides 44 (0.25 to 2μmol) were placed in an empty Twist synthesis column and 5 to 6 beads ofactivated 4 Å molecular sieves were introduced inside the column. Thecolumn was closed and flushed with argon followed by treatment with anoxidation solution.

The oxidation solution was prepared as follows: imidazole (150 mg, 2mmol, Aldrich) was coevaporated twice with anhydrous acetonitrile andthen dried under vacuum over P₂O₅. The residue was then redissolved inanhydrous acetonitrile (0.8 mL), anhydrous CCl₄ (0.8 mL, Aldrich),anhydrous triethylamine (0.1 mL, Sigma) and N,O-bis-trimethylsilylacetamide (0.4 mL, Aldrich). The resulting solution was dried overactivated 4 Å molecular sieves for 10 min, degassed with argon for 30seconds, and then pushed gently through the column for 5 hours at roomtemperature. The column was then emptied and quickly washed withmethanol (2×) followed by reverse flushed with argon to providephosphorimidazolidates 46.

D) Preparation of Solid Supported Diphosphate Oligonucleotides 48

1 mL of 0.5 M tris-tributylammonium phosphate (2 g in 8 mL of anhydrousDMF (prepared in-house) and dried over activated molecular sieves for 24h at 4° C., Aldrich) was pushed through the synthesis column for 30 h.The column was then emptied, washed several times with methanol andacetonitrile followed by reverse flushed with argon to provide thediphosphate oligonucleotides 48.

E) Cleavage of Diphosphate Oligonucleotides 48 from Solid Support

The dried solid support CPG carrying the diphosphate oligonucleotides 48were transferred from the Twist column to an empty screw cap plasticvial (10 mL). A solution of 30% NH₄OH (JTBaker)/ethanol (2 mL, 3:1 v/v)was added and left to react for 2 hours at room temperature. Thesolution was then decanted, evaporated and lyophilized from water toprovide the unbound diphosphate oligonucleotides 50.

Purification of the above oligonucleotides was performed via an Ionexchange semi-preparative chromatography followed by a semi-preparativereverse phase desalting using the AKTA purification system. Theconditions used were as follows: IEX-HPLC semi-prep: column: Dionex DNA;gradient (buffer A: 25 mM TRIZMA HCl (Aldrich); buffer B: 25 mM TRIZMAHC10.5 M NaClO₄ (Aldrich); 1 in 5 CV; flow 10 mL/min). RP-HPLCsemi-prep: column: C18; gradient (buffer A: 25 mM TEAB (Aldrich); bufferB: acetonitrile; 0 to 50% acetonitrile in 5 CV; flow 10 mL/min). Thedesalted fractions were collected, frozen, lyophilized and then storedat −20° C.

F) Analysis of Unbound Diphosphate Oligonucleotides 50 after Cleavagefrom Solid Support

The unbound oligonucleotides 50 were analyzed by ion exchange HPLC usinga gradient of 0 to 0.5 M NaClO₄ (25 mM TRIZMA) in 40 min on a DionexBioLC DNA Pac PA100 column. The samples were injected on a 25 OD per mLconcentration (10 μL injection) and the data was processed using Empower2 software. The molecular weight of the oligonucleotides was thendetermined after an LC/MS analysis on a RP LC-Q-T of mass spectrometer(Applied Biosystems) and ion deconvolution was applied for the molecularweight determination.

Example 22 Preparation of 5′ Diphosphate Oligonucleotides

A 40 μmol scale synthesis of the 5′ diphosphate oligonucleotide (A01)was performed on an ABI394 (Applied Biosystems) synthesizer and theunbound oligonucleotide after cleavage from the solid support wasanalyzed by ion exchange HPLC using the procedures illustrated inExample 21. The preparation and analysis of the oligonucleotide (A02) isalso carried out in the same manner as illustrated in Example 21.

SEQ ID NO./ OLIGO NO. Composition (5′ to 3′) 05/A01PP-T_(e)U_(f)G_(m)U_(f)C_(m)U_(f)C_(m)U_(f)G_(m)G_(f)U_(m)C_(f)C_(m)U_(f)U_(m)A_(f)C_(m)U_(f)U_(m)A_(ms)A_(m) 05/A02PP-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs)U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e)

A subscript “s” between two nucleosides indicates a phosphorothioateinternucleoside linkage. The absence of a subscript “s” between twonucleosides indicates a phosphodiester internucleoside linkage. A “PP”at the 5′-end indicates a 5′-diphosphate group. A nucleoside followed bya subscript “f” indicates a 2′-fluoro modified nucleoside, a nucleosidefollowed by a subscript “m” indicates a 2′-O-methyl modified nucleosideand a nucleoside followed by a subscript “e” indicates a2′-O-methoxyethyl (MOE) modified nucleoside.

Example 23 Modified ssRNAs Targeting PTEN In Vitro Study

A series of modified single strand RNAs (ssRNAs) were prepared andtested for their ability to reduce PTEN mRNA expression levels inhepatocyte cells. Hepatocytes were treated with the modified singlestranded oligomeric compounds shown below using LIPOFECTAMINE™ 2000 as atransfection reagent as described herein. The IC₅₀'s were calculatedusing the linear regression equation generated by plotting thenormalized mRNA levels to the log of the concentrations used and arepresented below.

SEQ ID NO./ OLIGO NO. Composition (5′ to 3′) 05/A02PP-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs)U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e) 05/447581P-T_(Rs)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs)U_(m)C_(fs)C_(m)U_(fs)U_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e) 05/456921P-T_(es)U_(fs)G_(m)U_(fs)C_(m)U_(fs)C_(m)U_(fs)G_(m)G_(fs)U_(m)C_(fs)C_(m)UfsU_(ms)A_(fs)C_(ms)U_(fs)U_(ms)A_(es)A_(e) 06/116847 ^(me)C_(es)T_(es)G_(es)^(me)C_(es)T_(es)A_(ds)G_(ds) ^(me)C_(ds) ^(me)C_(ds)T_(ds)^(me)C_(ds)T_(ds)G_(ds) G_(ds)A_(ds)T_(es)T_(es)T_(es)G_(es)A_(e)

A subscript “s” between two nucleosides indicates a phosphorothioateinternucleoside linkage. The absence of a subscript “s” between twonucleosides indicates a phosphodiester internucleoside linkage. A “PP”at the 5′-end indicates a 5′-diphosphate group. A “P” at the 5′-endindicates a 5′-monophosphate group. ^(me)C indicates a 5-methyl cytosinenucleoside. Nucleosides followed by a subscript “d” areβ-D-2′-deoxyribonucleosides. A nucleoside followed by a subscript “f”indicates a 2′-fluoro modified nucleoside, a nucleoside followed by asubscript “m” indicates a 2′-O-methyl modified nucleoside and anucleoside followed by a subscript “e” indicates a 2′-O-methoxyethyl(MOE) modified nucleoside.

Nucleosides followed by a subscript “R” have the structure shown below:

SEQ ID NO./ IC₅₀ OLIGO NO. Chemistry (nM) 05/A02 5′-diphosphate(P-O-P-O) 2 05/447581 5′-(R)-CH₃ monophosphate (P-O) 0.8 05/4569215′-monophosphate (P-O) 2 06/116847 5-10-5 MOE Gapmer 1

All publications, patents, and patent applications referenced herein areincorporated herein by reference. While in the foregoing specificationthis invention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

1-50. (canceled)
 51. A compound having Formula I:

wherein: Bx is a heterocyclic base moiety; each Pg is a hydroxylprotecting group; M₁ is H, OH or OR₁; M₂ is OH, OR₁ or N(R₁)(R₂); eachR₁ and R₂ is, independently, alkyl or substituted alkyl; r is 0 or 1; Ais O, S, CR₃R₄ or N(R₅); R₃ and R₄ are each, independently H, halogen,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; R₅ is H, C₁-C₆alkyl, substituted C₁-C₆ alkyl or a protecting group; Q₁ and Q₂ areeach, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl; G is H, OH, halogen or O—[C(R₆)(R₇)]_(n)-[(C═O)_(m)—X]_(j)—Z;each R₆ and R₇ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl; X is O, S or N(E₁); Z is H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃); E₁, E₂ and E₃ are each,independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl; n is from 1 toabout 6; m is 0 or 1; j is 0 or 1; each substituted group comprises oneor more optionally protected substituent groups independently selectedfrom H, halogen, OJ₁, N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁,OC(=L)N(J₁)(J₂), C(=L)N(J₁)(J₂), C(=L)N(H)—(CH₂)₂N(J₁)(J₂) or a mono orpoly cyclic ring system; L is O, S or NJ₃; each J₁, J₂ and J₃ is,independently, H or C₁-C₆ alkyl; when j is 1 then Z is other thanhalogen or N(E₂)(E₃); and when Q₁ and Q₂ are each H and G is H or OHthen A is other than O.
 52. The compound of claim 51 wherein Bx isuracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
 53. Thecompound of any one of claims 52 wherein r is 1, M₁ is H and M₂ is OH.54. The compound of any one of claims 53 wherein r is 0, M₁ is O(CH₂)₂CNand M₂ is N[CH(CH₃)₂]₂.
 55. The compound of claim 54 wherein G ishalogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃,OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃,O(CH₂)₃—N(R₆)(R₇), O(CH₂)₂—ON(R₆)(R₇), O(CH₂)₂—O(CH₂)₂—N(R₆)(R₇),OCH₂C(═O)—N(R₆)(R₇), OCH₂C(═O)—N(R₈)—(CH₂)₂—N(R₆)(R₇) orO(CH₂)₂—N(R₈)—C(═NR₉)[N(R₆)(R₇)] wherein R₆, R₇, R₈ and R₉ are each,independently, H or C₁-C₆ alkyl.
 56. The compound of claim 55 wherein Gis halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂.
 57. The compoundof claim 56 wherein G is F, OCH₃, O(CH₂)₂—OCH₃, OCH₂C(═O)—N(H)CH₃ orOCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂.
 58. The compound of claim 57 wherein G isO(CH₂)₂—OCH₃.
 59. The compound of claim 58 wherein each Pg is,independently, methyl, ethyl, isopropyl, tert-butyl, 2-cyanoethyl,benzyl, phenyl, 4-methoxybenzyl, 4-chlorobenzyl or 2-chlorophenyl. 60.The compound of claim 59 wherein Q₁ and Q₂ are each H.
 61. The compoundof claim 59 wherein one of Q₁ and Q₂ is H and the other of Q₁ and Q₂ isC₁-C₆ alkyl.
 62. The compound of claim 61 wherein the other of Q₁ and Q₂is CH₃.
 63. The compound of claim 60 wherein A is CR₃R₄.
 64. Thecompound of claim 63 wherein R₃ and R₄ are each H.
 65. The compound ofclaim 63 wherein one of R₃ and R₄ is H and the other of R₃ and R₄ isother than H.
 66. The compound of claim 65 wherein the other of R₃ andR₄ is F.
 67. The compound of claim 65 wherein the other of R₃ and R₄ isCH₃.
 68. The compound of claim 1 having the configuration of Formula Ia:


69. An oligomeric compound comprising a compound having Formula II:

wherein: Bx is a heterocyclic base moiety; T₁ is an internucleosidelinking group linking the compound of Formula II to the remainder of theoligomeric compound; each M₃ is, independently, H or a hydroxylprotecting group; A is O, S, CR₃R₄ or N(R₅); R₃ and R₄ are each,independently H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl; R₅ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or a protectinggroup; Q₁ and Q₂ are each, independently, H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl orsubstituted C₂-C₆ alkynyl; G is H, OH, halogen orO—[C(R₆)(R₇)]_(n)—[(C═O)_(m)—X]_(j)—Z; each R₆ and R₇ is, independently,H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl; X is O, S or N(E₁);Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃); E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl orsubstituted C₁-C₆ alkyl; n is from 1 to about 6; m is 0 or 1; j is 0 or1; each substituted group comprises one or more optionally protectedsubstituent groups independently selected from H, halogen, OJ₁,N(J₁)(J₂), ═NJ₁, SJ₁, N₃, CN, OC(=L)J₁, OC(=L)N(J₁)(J₂), C(=L)N(J₁)(J₂),C(=L)N(H)—(CH₂)₂N(J₁)(J₂) or a mono or poly cyclic ring system; L is O,S or NJ₃; each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; when jis 1 then Z is other than halogen or N(E₂)(E₃); and when Q₁ and Q₂ areeach H and G is H or OH then A is other than O.
 70. The oligomericcompound of claim 69 wherein Bx is uracil, thymine, cytosine,5-methylcytosine, adenine or guanine.